Methods for treating hypophosphatemic disorders

ABSTRACT

The present invention provides compositions and methods for treating a hypophosphatemic disorder, such as X-linked hypophosphatemia (XLH). The method entails administering to a subject a pharmaceutical composition containing an anti-FGF23 ligand, wherein the dosing regimen of the pharmaceutical is designed to reach effective and efficient control of FGF23 activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. application Ser.No. 14/725,320, filed May 29, 2015, which claims priority to U.S.Provisional Application Ser. No. 62/009,474, filed Jun. 9, 2014, whichis herein incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to compositions comprising an activeingredient regulating bone formation, and methods of using thecompositions.

DESCRIPTION OF TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:ULPI_022_02US_SeqList_ST25.txt, date created: Mar. 26, 2020, file size:28,459 bytes).

BACKGROUND OF THE INVENTION

Fibroblast growth factor-23 (FGF23 or FGF-23) is a hormone thatregulates phosphate level through an action on the reabsorption ofphosphate from the kidneys. FGF23 was cloned initially from mice usingPCR methods based on sequences derived from database searches usingsequence homology with FGF15. Human FGF23 was cloned by using sequencehomology with mouse FGF23 (Yamashita, T. et al., Biochem. Biophy. Res.Commun., 277: 494-498, 2000).

Recent studies have shed new light on the understanding of phosphatemetabolism. Phosphate has important functions in the body and severalmechanisms have evolved to regulate phosphate balance including vitaminD, parathyroid hormone and phosphatonins such as FGF23. Disorders ofphosphate homeostasis leading to hypo- and hyperphosphataemia are commonand have clinical and biochemical consequences.

Despite the previous findings, there remains a need for more effectivemethods for treating disorders related to abnormal phosphate metabolism,such as disorders related to abnormal FGF23 signaling, and methods forevaluating the efficacy of the treatment. The present invention meetsthis need and provides compositions and methods for the effectivetreatment of such disorders.

SUMMARY OF THE INVENTION

The present invention is based in part on the discovery that dosingregimen of anti-FGF23 ligands can be determined based on anunderstanding of the relationship between phosphate levels and regulatedlevels of FGF23 within a dosing cycle during chronic administration.Accordingly, the present invention provides methods for determining thedosing regimen of anti-FGF23 ligands. The present invention alsoprovides methods for treating conditions or disorders associated withFGF23. Anti-FGF23 ligands can be used for treating various disorders. Insome other embodiments, the disorders are associated with abnormal FGF23signaling in a subject in need of such treatment. In some otherembodiments, the disorders are associated with abnormal FGF23 activityin a subject in need of such treatment. In some other embodiments, thedisorders are associated with abnormally high FGF23 activity in asubject in need of such treatment. In some other embodiments, thedisorder is selected from the group consisting of, autosomal dominanthypophosphatemic rickets/osteomalachia (ADHR), X-linked hypophosphatemia(XLH), autosomal recessive hypophosphatemic rickets (ARHR), fibrousdysplasia (FD), McCune-Albright syndrome complicated by fibrousdysplasia (MAS/FD), Jansen's metaphyseal chondrodysplasia (Jansen'sSyndrome), autosomal dominant polycystic kidney disease (ADPKD),tumor-induced osteomalacia (TIO), chronic metabolic acidosis and ectopiccalcification. In some embodiments, the disorder is XLH. In someembodiments, the methods comprise administering an anti-FGF23 ligand toa subject in need of such treatment. In some embodiments, an effectiveamount of an anti-FGF23 ligand is administered to the subject.

According to one aspect of the present invention, the dosing regimen ofthe anti-FGF23 ligand can be determined based on one or more PDparameters of the anti-FGF23 ligand in the subject.

The PD parameters of the anti-FGF23 ligand include but are not limitedto, serum phosphorus (e.g., AUC serum phosphorus, such as AUC serumphosphorus within a dose interval, or peak and trough serum phosphorus),Tmp/GFR, serum 1,25-dihydroxy vitamin D, and serum 25-hydroxy vitamin D.

In some embodiments, the anti-FGF23 ligand is selected from the groupconsisting of anti-FGF23 antibodies, FGF23 antisense oligonucleotides,small molecule inhibitors of FGF23, FGF23 antagonists, and combinationsthereof. In some embodiments, the anti-FGF23 ligand is an anti-FGF23antibody.

In some embodiments, the anti-FGF23 ligand comprises one or more CDRsequences selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO. 3, SEQ IDNO: 4, SEQ ID NO: 5, and SEQ ID NO. 6.

In some embodiments, the dosing regimen of the anti-FGF23 ligandprovides an adequate increase or predetermined production of phosphateover a dosing cycle. In some embodiments, the dosing regimen provides astable increase of phosphate within a predetermined range of phosphate.In some embodiments, the dosing regimen provides a stable increase inserum phosphorus of about 0.5 to about 1.5 mg/dL above the baselinelevel of phosphate.

In some embodiments, the dosing regimen of the anti-FGF23 ligandprovides sufficient binding agent for an increased bound FGF23 levelduring chronic anti-FGF23 ligand therapy.

In some embodiments, the dosing regimen comprises a dosing frequencythat provides a sustained effect on the NaPi transporter, i.e., withouta decline of NaPi transporter activity during a dosing cycle.

In some embodiments, the dosing regimen comprises administering ananti-FGF23 ligand more frequently than a monthly dosing regimen, such asadministering an anti-FGF23 ligand every two weeks (i.e., Q2W).

In some embodiments, the dosing regimen provides an increase in boneremodeling.

In yet some other embodiments, the present invention provides methods oftreating and/or preventing a disorder by administering about every twoweeks an effective amount of an anti-FGF23 ligand to a subject in needof such a treatment. In some embodiments, the administration providesstatistically significant therapeutic effect for treating the disorder.In some embodiments, the subject has a disorder selected from the groupconsisting of, autosomal dominant hypophosphatemic rickets/osteomalacia(ADHR), X-linked hypophosphatemia (XLH), autosomal recessivehypophosphatemic rickets (ARHR), fibrous dysplasia (FD), McCune-Albrightsyndrome complicated by fibrous dysplasia (MAS/FD), Jansen's metaphysealchondrodysplasia (Jansen's Syndrome), autosomal dominant polycystickidney disease (ADPKD), tumor-induced osteomalacia (TIO), chronicmetabolic acidosis, and ectopic calcification. In some embodiments, thedisorder is ectopic calcification.

According to another aspect of the invention, it provides methods oftreating conditions or disorders associated with FGF23. In someembodiments, the methods comprise administering one or more anti-FGF23ligands according to a dosing regimen determined based on one or more PDparameters of the anti-FGF23 ligands.

According to yet another aspect of the invention, it also providesmethods of increasing bone remodeling. In some embodiments, the methodscomprise administering to a subject in need of such treatment aneffective amount of an anti-FGF23 ligand. In some embodiments, theanti-FGF23 ligand causes an increase of a marker. In some embodiments,the marker is selected from the group consisting of serum Type 1Pro-collagen/N-terminal (P1NP), carboxy-terminal collagen crosslink(CTX), Osteocalcin, BALP, and serum CTx and urine NTX/creatine ratio. Insome embodiments, the subject has a disorder selected from the groupconsisting of, autosomal dominant hypophosphatemic rickets/osteomalachia(ADHR), X-linked hypophosphatemia (XLH), autosomal recessivehypophosphatemic rickets (ARHR), fibrous dysplasia (FD), McCune-Albrightsyndrome complicated by fibrous dysplasia (MAS/FD), Jansen's metaphysealchondrodysplasia (Jansen's Syndrome), autosomal dominant polycystickidney disease (ADPKD), tumor-induced osteomalacia (TIO), chronicmetabolic acidosis, and ectopic calcification. In some embodiments, thesubject has XLH. In some other embodiments, the method increases boneremodeling associated with one or more conditions caused by abnormalFGF23, e.g., high FGF23 activity and/or level.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts mean (±SD) serum phosphorus values by study day for allsubjects treated with KRN23 in the efficacy analysis during the firstPhase I/II clinical trial.

FIG. 2 depicts mean (±SD) TmP/GFR levels by study day for all subjectstreated with KRN23 in the efficacy analysis during the first Phase I/IIclinical trial.

FIGS. 3A and 3B depicts scatter plots of AUC_(last) (FIG. 3A) andAUC_(n) (FIG. 3B) for serum phosphorus versus AUC_(last) (FIG. 3A) andAUC_(n) (FIG. 3B) for TmP/GFR in patients treated with KRN23 in theefficacy analysis during the first Phase I/II clinical trial.

FIG. 4 depicts scatter plot of TmP/GFR calculated versus TmP/GFRmanually read from Nomogram for all subjects treated with KRN23 in theefficacy analysis during the first Phase I/II clinical trial.

FIG. 5 depicts mean (±SD) 1,25(OH)₂D Levels (pg/mL) over time for the 4dosing intervals for all subjects treated with KRN23 in the efficacyanalysis during the first Phase I/II clinical trial.

FIG. 6 depicts mean (±SD) total FGF23 and unbound FGF23 values over timein the efficacy analysis during the first Phase I/II clinical trial.

FIGS. 7A and 7B depict the mean (±SD) KRN23 concentration over time(during the 4 dosing Intervals) for the pharmacokinetic analysis duringthe first Phase I/II clinical trial.

FIGS. 8A and 8B depict a scatter plot of AUC for serum phosphoruschanges from baseline versus serum KRN23 AUC in the efficacy analysisduring the first Phase I/II clinical trial.

FIGS. 9A and 9B depict scatter plots of AUC for TMP/GFR change frombaseline versus serum KRN23 AUC in a pharmacokinetic analysis during thefirst Phase I/II clinical trial.

FIGS. 10A and 10B depict scatter plots of AUC for serum1,25-Dihydroxyvitamin D change from baseline versus serum KRN23 AUC inpharmacokinetic analysis during the first Phase I/II clinical trial.

FIG. 11 depicts relationship between KRN23 concentration and change frombaseline in serum phosphorus in adult subjects with XLH treated withKRN23 from the population PK-PD model predictions in pharmacokineticanalysis during the first Phase I/II clinical trial.

FIG. 12 depicts mean (±SD) serum phosphorus values over time—allsubjects treated with KRN23 for efficacy analysis during the secondPhase I/II clinical trial.

FIG. 13 depicts mean (±SD) TmP/GFR levels over time of all subjectstreated with KRN23 for efficacy analysis during the second Phase I/IIclinical trial.

FIG. 14 depicts mean (±SD) 1,25(OH)₂D levels over time of all subjectstreated with KRN23 for efficacy analysis during the second Phase I/IIclinical trial.

FIG. 15 depicts mean (±SD) total intact FGF23 values over time forefficacy analysis during the second Phase I/II clinical trial.

FIG. 16 depicts mean (±SD) total unbound intact FGF23 values over timefor efficacy analysis during the second Phase I/II clinical trial.

FIG. 17 depicts proposed model of X-linked hypophosphatemiapathophysiology, and treatment by the anti-FGF23 antibody KRN23.

FIG. 18 depicts a schematic of the proposed Phase 2 study design.

FIG. 19 depicts serum phosphorus levels in pediatric patients treatedwith a Q2W dosing regimen of the anti-FGF23 antibody KRN23.

FIG. 20 depicts serum phosphorus levels in pediatric patients treatedwith a Q4W dosing regimen of the anti-FGF23 antibody KRN23.

FIG. 21 depicts a side-by-side comparison of serum phosphorus levels inpediatric patients treated with the anti-FGF23 antibody KRN23 in a Q2Wdosing regimen or a Q4W dosing regimen.

FIG. 22 depicts TmP/GRF levels in pediatric patients treated with a Q2Wdosing regimen of the anti-FGF23 antibody KRN23.

FIG. 23 depicts TmP/GRF levels in pediatric patients treated with a Q4Wdosing regimen of the anti-FGF23 antibody KRN23.

FIG. 24 depicts a side-by-side comparison of TmP/GRF levels in pediatricpatients treated with the anti-FGF23 antibody KRN23 in a Q2W dosingregimen or a Q4W dosing regimen.

FIG. 25 depicts a side-by-side comparison of serum alkaline phosphatase(ALP) levels in pediatric patients treated with the anti-FGF23 antibodyKRN23 in a Q2W dosing regimen or a Q4W dosing regimen.

DETAILED DESCRIPTION Definitions

The verb “comprise” as is used in this description and in the claims andits conjugations are used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded.

The term “a” or “an” refers to one or more of that entity; for example,“a gene” refers to one or more genes or at least one gene. As such, theterms “a” (or “an”), “one or more” and “at least one” are usedinterchangeably herein. In addition, reference to “an element” by theindefinite article “a” or “an” does not exclude the possibility thatmore than one of the elements are present, unless the context clearlyrequires that there is one and only one of the elements.

The invention provides isolated, chimeric, recombinant or syntheticpolynucleotide sequences. As used herein, the terms “polynucleotide”,“polynucleotide sequence”, “nucleic acid sequence”, “nucleic acidfragment”, and “isolated nucleic acid fragment” are used interchangeablyherein and encompass DNA, RNA, cDNA, whether single stranded or doublestranded, as well as chemical modifications thereof. These termsencompass nucleotide sequences and the like. A polynucleotide may be apolymer of RNA or DNA that is single- or double-stranded, thatoptionally contains synthetic, non-natural or altered nucleotide bases.A polynucleotide in the form of a polymer of DNA may be comprised of oneor more segments of cDNA, genomic DNA, synthetic DNA, or mixturesthereof. Nucleotides (usually found in their 5′-monophosphate form) arereferred to by a single letter designation as follows: “A” for adenylateor deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate ordeoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate,“T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines(C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N”for any nucleotide. In some embodiments, the isolated, chimeric,recombinant or synthetic polynucleotide sequences are derived from genemarkers of the present invention.

The invention also provides proteins or polypeptides. In someembodiments the proteins or polypeptides are isolated, purified,chimeric, recombinant or synthetic. As used herein, the term“polypeptide” or “protein” refers to amino acid polymers of any length.The polymer may be linear or branched, it may comprise modified aminoacids, and it may be interrupted by non-amino acids. The terms alsoencompass an amino acid polymer that has been modified naturally or byintervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labelling component. Alsoincluded are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),as well as other modifications known in the art. Polypeptides can occuras single chains or associated chains. Polypeptides of the invention cantake various forms (e.g. native, fusions, glycosylated,non-glycosylated, lipidated, non-lipidated, phosphorylated,non-phosphorylated, myristoylated, non-myristoylated, monomeric,multimeric, particulate, denatured, etc.). In some embodiments, thesequences of the proteins or polypeptides are derived from gene markersof the present invention.

Single letter amino acid abbreviations used herein have their standardmeaning in the art, and all peptide sequences described herein arewritten according to convention, with the N-terminal end to the left andthe C-terminal end to the right.

As used herein, the term “a component in the FGF23 signaling pathway”,refers to FGF23, or other components that can modulate the activity ofFGF23, directly or indirectly, or components that can be modulated byFGF23, directly or indirectly. Such components include, but are notlimited to those described by Sapir-Koren and Livshits (Bonemineralization and regulation of phosphate homeostasis, IBMS BoneKEy,8:286-300, (2011)), Quarles (FGF23, PHEX, and MEPE regulation ofphosphate homeostasis and skeletal mineralization, American Journal ofPhysiology—Endocrinology and Metabolism Published 1 Jul. 2003, Vol. 285,no. E1-E9), and Martin and Quarles (Evidence for Fgf23 Involvement in aBone—Kidney Axis Regulating Bone Mineralization and Systemic Phosphateand Vitamin D Homeostasis, Endocrine FGFs and Klothos, Springer Scienceand Business Media, LLC, landers Bioscience), each of which is hereinincorporated by reference in its entirety for all purposes.

As used herein, the term “fibroblast growth factor receptor” or “FGFR”refers to a receptor specific for FGF which is necessary for transducingthe signal exerted by FGF to the cell interior, typically comprising anextracellular ligand-binding domain, a single transmembrane helix, and acytoplasmic domain having tyrosine kinase activity. The FGFRextracellular domain consists of three immunoglobulin-like (Ig-like)domains (D1, D2 and D3), a heparin binding domain and an acidic box.

The term “antigen,” as used herein, refers to a molecule capable ofbeing recognized by an antibody. An antigen can be, for example, apeptide or a modified form thereof. An antigen can comprise one or moreepitopes.

The term “epitope,” as used herein, is a portion of an antigen that isspecifically recognized by an antibody. An epitope, for example, cancomprise or consist of a portion of a peptide (e.g., a peptide of theinvention). An epitope can be a linear epitope, sequential epitope, or aconformational epitope.

As used herein, the term “anti-FGF23 ligand” refers to molecules thatinhibit the activity of FGF23 directly or indirectly. The inhibition canhappen at the DNA level, transcriptional level, post-transcriptionallevel, translational level, and/or post-translational level. Suchmolecules include, but are not limited to, anti-FGF23 antibodies, FGF23antisense oligonucleotides, small molecule inhibitors of FGF23, FGF23antagonists as well as any molecule that interacts with one or morecomponents of FGF23 signaling pathway, thereby indirectly inhibitingFGF23.

The terms “antibody” and “immunoglobulin”, as used herein, refer broadlyto any immunological binding agent or molecule that comprises a humanantigen binding domain, including polyclonal and monoclonal antibodies.Depending on the type of constant domain in the heavy chains, wholeantibodies are assigned to one of five major classes: IgA, IgD, IgE,IgG, and IgM and the antibodies of the invention may be in any one ofthese classes. Several of these are further divided into subclasses orisotypes, such as IgG1, IgG2, IgG3, IgG4, and the like. The heavy-chainconstant domains that correspond to the difference classes ofimmunoglobulins are termed α, δ, ε, μ, and respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known. In some embodiments, IgG and/or IgM areused. It should be understood that when the terms “antibody” or“antibodies” are used, this is intended to include intact antibodies,such as polyclonal antibodies or monoclonal antibodies (mAbs), as wellas proteolytic fragments thereof such as the Fab or F(ab′)2 fragments.Further included within the scope of the invention are chimericantibodies; human and humanized antibodies; recombinant and engineeredantibodies, and fragments thereof. Furthermore, the DNA encoding thevariable region of the antibody can be inserted into the DNA encodingother antibodies to produce chimeric antibodies (see, for example, U.S.Pat. No. 4,816,567). Single chain antibodies fall within the scope ofthe present invention.

By the term “single chain variable fragment (scFv)” is meant a fusion ofthe variable regions of the heavy and light chains of immunoglobulin,linked together with a short (usually serine, glycine) linker. Singlechain antibodies can be single chain composite polypeptides havingantigen binding capabilities and comprising amino acid sequenceshomologous or analogous to the variable regions of an immunoglobulinlight and heavy chain (linked VH-VL or single chain Fv (scFv)). Both VHand VL may copy natural monoclonal antibody sequences or one or both ofthe chains may comprise a CDR-FR construct of the type described in U.S.Pat. No. 5,091,513, the entire contents of which are incorporated hereinby reference. The separate polypeptides analogous to the variableregions of the light and heavy chains are held together by a polypeptidelinker. Methods of production of such single chain antibodies,particularly where the DNA encoding the polypeptide structures of the VHand VL chains are known, may be accomplished in accordance with themethods described, for example, in U.S. Pat. Nos. 4,946,778, 5,091,513and 5,096,815, the entire contents of each of which are incorporatedherein by reference.

The term “light chain” includes a full-length light chain and fragmentsthereof having sufficient variable region sequence to confer bindingspecificity. A full-length light chain includes a variable regiondomain, VL, and a constant region domain, CL. The variable region domainof the light chain is at the amino-terminus of the polypeptide. Lightchains include kappa chains and lambda chains.

The term “heavy chain” includes a full-length heavy chain and fragmentsthereof having sufficient variable region sequence to confer bindingspecificity. A full-length heavy chain includes a variable regiondomain, V_(H), and three constant region domains, C_(H)L C_(H)2, andC_(H)3. The V_(H) domain is at the amino-terminus of the polypeptide,and the C_(H) domains are at the carboxyl-terminus, with the C_(H)3being closest to the carboxy-terminus of the polypeptide. Heavy chainscan be of any isotype, including IgG (including IgG1, IgG2, IgG3 andIgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.

Three complementarity determining regions are present in the heavy chainvariable region, which are a first complementarity determining region(CDR1), a second complementarity determining region (CDR2), and a thirdcomplementarity determining region (CDR3). The three complementaritydetermining regions in the heavy chain variable region are collectivelyreferred to as the heavy chain complementarity determining region.Similarly, three complementarity determining regions are present in thelight chain variable region, which are a first complementaritydetermining region (CDR1), a second complementarity determining region(CDR2), and a third complementarity determining region (CDR3). The threecomplementarity determining regions in the light chain variable regionare collectively referred to as the light chain complementaritydetermining region. The sequences of these CDRs can be determined byusing the methods described in Sequences of Proteins of ImmunologicalInterest, US Dept. Health and Human Services (1991) and the like.

The term “immunologically functional fragment” (or simply “fragment”) ofan antigen binding protein, e.g., an antibody or immunoglobulin chain(heavy or light chain), as used herein, is an antigen binding proteincomprising a portion (regardless of how that portion is obtained orsynthesized) of an antibody that lacks at least some of the amino acidspresent in a full-length chain but which is capable of specificallybinding to an antigen. Such fragments are biologically active in thatthey bind specifically to the target antigen and can compete with otherantigen binding proteins, including intact antibodies, for specificbinding to a given epitope. In one aspect, such a fragment will retainat least one CDR present in the full-length light or heavy chain, and insome embodiments will comprise a single heavy chain and/or light chainor portion thereof. These biologically active fragments can be producedby recombinant DNA techniques, or can be produced by enzymatic orchemical cleavage of antigen binding proteins, including intactantibodies. Immunologically functional immunoglobulin fragments include,but are not limited to, Fab, Fab′, F(ab′)2, Fv, domain antibodies andsingle-chain antibodies, and can be derived from any mammalian source,including but not limited to human, mouse, rat, camelid or rabbit. It iscontemplated further that a functional portion of the antigen bindingproteins disclosed herein, for example, one or more CDRs, could becovalently bound to a second protein or to a small molecule to create atherapeutic agent directed to a particular target in the body,possessing bifunctional therapeutic properties, or having a prolongedserum half-life.

An “Fc” or “Fc region” comprises one or two heavy chain fragments, andcan comprise the C_(H)2 and/or C_(H)3 domains of an antibody. When twoheavy chain fragments are present, the two heavy chain fragments areheld together by two or more disulfide bonds and by hydrophobicinteractions of the C_(H)3 domains. An Fc region can be naturallyoccurring (e.g., a Fc region derived from an IgG1, IgG2, IgG3, IgG4,IgE, IgA, etc.) or can be an engineered sequence comprising one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, etc.) mutations, deletions or insertions introduced into anaturally occurring heavy chain fragment or fragments that make up an Fcsequence.

An “Fab′ fragment” contains one light chain and a portion of one heavychain that contains the VH domain and the C_(H)1 domain and also theregion between the C_(H)1 and C_(H)2 domains, such that an interchaindisulfide bond can be formed between the two heavy chains of two Fab′fragments to form an F(ab′)₂ molecule.

An “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H)1 andC_(H)2 domains, such that an interchain disulfide bond is formed betweenthe two heavy chains. A F(ab′)₂ fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains.

The “Fv region” comprises the variable regions from both the heavy andlight chains, but lacks the constant regions.

As will be understood by those in the art, the immunological bindingreagents encompassed by the term “antibody” extend to all antibodies andantigen binding fragments thereof, including whole antibodies, dimeric,trimeric and multimeric antibodies; bispecific antibodies; chimericantibodies; recombinant and engineered antibodies, and fragmentsthereof. The term “antibody” is thus used to refer to any antibody-likemolecule that has an antigen binding region, and this term includesantibody fragments that comprise an antigen binding domain such as Fab′,Fab, F(ab′)2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv(single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies,diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fabfusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); Bispecific T-cell Engager (BiTE)(scFv-scFv tandems to attract T cells); dual variable domain (DVD)-Ig(bispecific format); small immunoprotein (SIP) (kind of minibody); SMIP(“small modular immunopharmaceutical” scFv-Fc dimer; DART (ds-stabilizeddiabody “Dual Affinity ReTargeting”); small antibody mimetics comprisingone or more CDRs and the like. The techniques for preparing and usingvarious antibody-based constructs and fragments are well known in theart (see Kabat et al., 1991, specifically incorporated herein byreference). Diabodies, in particular, are further described in EP 404,097 and WO 93/11161; whereas linear antibodies are further described inZapata et al. (1995).

Antibodies can be fragmented using conventional techniques. For example,F(ab′)2 fragments can be generated by treating the antibody with pepsin.The resulting F(ab′)2 fragment can be treated to reduce disulfidebridges to produce Fab′ fragments. Papain digestion can lead to theformation of Fab fragments. Fab, Fab′ and F(ab′)2, scFv, Fv, dsFv, Fd,dAbs, T and Abs, ds-scFv, dimers, minibodies, diabodies, bispecificantibody fragments and other fragments can also be synthesized byrecombinant techniques or can be chemically synthesized. Techniques forproducing antibody fragments are well known and described in the art.For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; LeGall et al., 2004; Reff & Heard, 2001; Reiter et al., 1996; and Young etal., 1995 further describe and enable the production of effectiveantibody fragments.

The antibodies or antibody fragments can be produced naturally or can bewholly or partially synthetically produced. Thus the antibody may befrom any appropriate source, for example recombinant sources and/orproduced in transgenic animals or transgenic plants, or in eggs usingthe IgY technology. Thus, the antibody molecules can be produced invitro or in vivo. In some embodiments, the antibody or antibody fragmentcomprises an antibody light chain variable region (VL) that comprisesthree CDR domains and an antibody heavy chain variable region (VH) thatcomprises three CDR domains. Said VL and VH generally form the antigenbinding site.

As used herein, an “Fv” fragment is the minimum antibody fragment thatcontains a complete antigen-recognition and binding site. This regionhas a dimer of one heavy chain and one light chain variable domain intight, non-covalent association. It is in this configuration that thethree hypervariable regions (CDRs) of each variable domain interact todefine an antigen-binding site on the surface of the VH-VL dimer.Collectively, the six hypervariable regions (CDRs) conferantigen-binding specificity to the antibody. However, it is welldocumented in the art that the presence of three CDRs from the lightchain variable domain and three CDRs from the heavy chain variabledomain of an antibody is not necessary for antigen binding. Thus,constructs smaller than the above classical antibody fragment are knownto be effective. For example, camelid antibodies (Hamers-Casterman etal., 1993; Arbabi Ghahroudi et al., 1997) have an extensive antigenbinding repertoire but are devoid of light chains. Also, results withsingle domain antibodies comprising VH domains alone (Ward et al., 1989;Davies and Riechmann, 1995) or VL domains alone (van den Beucken et al.,2001) show that these domains can bind to antigen with acceptably highaffinities. Thus, three CDRs can effectively bind antigen.

It is also known that a single CDR, or two CDRs, can effectively bindantigen. As a first example, a single CDR can be inserted into aheterologous protein and confer antigen binding ability on theheterologous protein, as exemplified by showing that a VH CDR3 regioninserted into a heterologous protein, such as GFP, confers antigenbinding ability on the heterologous protein (Kiss et al., 2006; Nicaiseet al., 2004). It is further known that two CDRs can effectively bindantigen, and even confer superior properties than possessed by theparent antibody. For example, it has been shown (Qiu et al., 2007) thattwo CDRs from a parent antibody (a VH CDR1 and a VL CDR3 region) retainthe antigen recognition properties of the parent molecule but have asuperior capacity to penetrate tumors. Joining these CDR domains with anappropriate linker sequence (e.g., from VH FR2) to orientate the CDRs ina manner resembling the native parent antibody produced even betterantigen recognition. Therefore, it is known in the art that it ispossible to construct antigen binding antibody mimetics comprising twoCDR domains (e.g., one from a VH domain and one from a VL domain, forexample, with one of the two CDR domains being a CDR3 domain) orientatedby means of an appropriate framework region to maintain the conformationfound in the parent antibody. Thus, although some antibodies of theinvention might comprise six CDR regions (three from a light chain andthree from a heavy chain), antibodies with fewer than six CDR regionsand as few as one or two CDR regions are encompassed by the invention.In addition, antibodies with CDRs from only the heavy chain or lightchain are also contemplated.

Light chain CDR regions for use in conjunction with the specified heavychain CDR regions are described elsewhere herein. However, other lightchain variable regions that comprise three CDRs for use in conjunctionwith the heavy chain variable regions of the invention are alsocontemplated. Appropriate light chain variable regions which can be usedin combination with the heavy chain variable regions of the inventionand which give rise to an antibody which binds FGF23 can be readilyidentified by a person skilled in the art. For example, a heavy chainvariable region of the invention can be combined with a single lightchain variable region or a repertoire of light chain variable regionsand the resulting antibodies tested for binding to FGF23. It would beexpected that a reasonable number of such combinations of heavy chainvariable regions of the invention with different light chain variableregions would retain the ability to bind FGF23.

Similar methods could be used to identify alternative heavy chainvariable regions for use in combination with light chain variableregions of the invention. In certain embodiments, the antibody orantibody fragment comprises all or a portion of a heavy chain constantregion, such as an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, IgM or IgDconstant region. In some embodiments, the heavy chain constant region isan IgG1 heavy chain constant region, or a portion thereof. Furthermore,the antibody or antibody fragment can comprise all or a portion of akappa light chain constant region or a lambda light chain constantregion, or a portion thereof. All or part of such constant regions maybe produced naturally or may be wholly or partially synthetic.Appropriate sequences for such constant regions are well known anddocumented in the art. When a full complement of constant regions fromthe heavy and light chains are included in the antibodies of theinvention, such antibodies are typically referred to herein as “fulllength” antibodies or “whole” antibodies.

“Functional fragment” is a portion of an antibody (partial fragment) andhas one or more of the actions of the antibody to the antigen. In otherwords, it refers to a fragment which retains binding ability to theantigen, reactivity to the antigen, or recognition capability to theantigen. Examples include Fv, disulfide stabilized Fv (dsFv), singlechain Fv (scFv), and polymers of these and the like. Stated morespecifically, examples include peptides which contain Fab, Fab′, F(ab′)₂, scFv, diabody, dsFv, and CDR (D. J. King., Applications andEngineering of Monoclonal Antibodies., 1998 T. J. International Ltd).

The antibodies of the present invention also includes derivatives of theantibody, such as those derivatives in which radioisotopes, lowmolecular weight drugs, macromolecular drugs, proteins, and the like isbound chemically or through genetic engineering to the antibody againstFGF23 of the present invention or functional fragments of the antibody.The derivatives of the antibody of the present invention can be producedby bonding radioisotopes, low molecular weight drugs, macromoleculardrugs, proteins and the like to the amino terminal side or carboxyterminal side of the H chain (heavy chain) or L chain (light chain) ofthe antibody against FGF23 of the present invention or the functionalfragment of the antibody, to a suitable substituted group or side chainin the antibody or functional fragment of the antibody, and further, toa sugar chain in the antibody or functional fragment of the antibody andthe like by chemical methods (Koutai Kogaku Nyuumon, Osamu Kanamitsu,Chijin Shokan, 1994) and the like. In addition, the derivative of theantibody bonded with protein is produced by linking the DNA whichencodes the antibody against FGF23 of the present invention and thefunctional fragment of the antibody and the DNA which encodes theprotein to be bonded, inserting this DNA into an expression vector, andintroducing and expressing the expression vector in a suitable hostcell.

In the present invention, “human antibody” is defined as an antibodywhich is an expression product of an antibody gene derived from humans.Human antibody, as will be described later, can be obtained byintroducing the human antibody gene locus and by administering antigento transgenic animals having the ability to produce human antibody.Examples of these transgenic animals include mice. The method ofcreation of mice which can produce human antibody is described, forexample, in International Publication Number WO 2002/43478.

A “variant” of a polypeptide comprises an amino acid sequence whereinone or more amino acid residues are inserted into, deleted from and/orsubstituted into the amino acid sequence relative to another polypeptidesequence. Variants include fusion proteins.

A “derivative” of a polypeptide is a polypeptide that has beenchemically modified in some manner distinct from insertion, deletion, orsubstitution variants, e.g., via conjugation to another chemical moiety.

The term “naturally occurring” as used throughout the specification inconnection with biological materials such as polypeptides, nucleicacids, host cells, and the like, refers to materials which are found innature.

The term “identity” refers to a relationship between the sequences oftwo or more polypeptide molecules or two or more nucleic acid molecules,as determined by aligning and comparing the sequences. “Percentidentity” means the percent of identical residues between the aminoacids or nucleotides in the compared molecules and is calculated basedon the size of the smallest of the molecules being compared. For thesecalculations, gaps in alignments (if any) must be addressed by aparticular mathematical model or computer program (i.e., an“algorithm”). Methods that can be used to calculate the identity of thealigned nucleic acids or polypeptides include those described inComputational Molecular Biology, (Lesk, A. M., ed.), (1988) New York:Oxford University Press; Biocomputing Informatics and Genome Projects,(Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysisof Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.),1994, New Jersey: Humana Press; von Heinje, G., (1987) Sequence Analysisin Molecular Biology, New York: Academic Press; Sequence AnalysisPrimer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M.Stockton Press; and Carillo et al., (1988) SIAM I Applied Math. 48:1073.

In calculating percent identity, the sequences being compared arealigned in a way that gives the largest match between the sequences. Thecomputer program used to determine percent identity can be, for example,the GCG program package, which includes GAP (Devereux et al., (1984)Nucl. Acid Res. 12:387; Genetics Computer Group, University ofWisconsin, Madison, Wis.). The computer algorithm GAP is used to alignthe two polypeptides or polynucleotides for which the percent sequenceidentity is to be determined. The sequences are aligned for optimalmatching of their respective amino acid or nucleotide (the “matchedspan”, as determined by the algorithm). A gap opening penalty (which iscalculated as 3× the average diagonal, wherein the “average diagonal” isthe average of the diagonal of the comparison matrix being used; the“diagonal” is the score or number assigned to each perfect amino acidmatch by the particular comparison matrix) and a gap extension penalty(which is usually 1/10 times the gap opening penalty), as well as acomparison matrix such as PAM 250 or BLOSUM 62 are used in conjunctionwith the algorithm. In certain embodiments, a standard comparison matrix(see, Dayhoff et al., (1978) Atlas of Protein Sequence and Structure5:345-352 for the PAM 250 comparison matrix; Henikoff et al., (1992)Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62comparison matrix) is also used by the algorithm.

Recommended parameters for determining percent identity for polypeptidesor nucleotide sequences using the GAP program are the following:

Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453;

Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra;

Gap Penalty: 12 (but with no penalty for end gaps)

Gap Length Penalty: 4

Threshold of Similarity: 0

Certain alignment schemes for aligning two amino acid sequences canresult in matching of only a short region of the two sequences, and thissmall aligned region can have very high sequence identity even thoughthere is no significant relationship between the two full-lengthsequences. Accordingly, the selected alignment method (e.g., the GAPprogram) can be adjusted if so desired to result in an alignment thatspans at least 50 contiguous amino acids of the target polypeptide.

The terms “treating” and “treatment” as used herein refer to an approachfor obtaining beneficial or desired results including clinical results,and may include even minimal changes or improvements in one or moremeasurable markers of the disease or condition being treated. Atreatment is usually effective to reduce at least one symptom of acondition, disease, disorder, injury or damage. Exemplary markers ofclinical improvement will be apparent to persons skilled in the art.Examples include, but are not limited to, one or more of the following:decreasing the severity and/or frequency one or more symptoms resultingfrom the disease, diminishing the extent of the disease, stabilizing thedisease (e.g., preventing or delaying the worsening of the disease),delay or slowing the progression of the disease, ameliorating thedisease state, decreasing the dose of one or more other medicationsrequired to treat the disease, and/or increasing the quality of life,etc.

“Prophylaxis,” “prophylactic treatment,” or “preventive treatment”refers to preventing or reducing the occurrence or severity of one ormore symptoms and/or their underlying cause, for example, prevention ofa disease or condition in a subject susceptible to developing a diseaseor condition (e.g., at a higher risk, as a result of geneticpredisposition, environmental factors, predisposing diseases ordisorders, or the like).

The term “disorder” or “disease” used interchangeably herein, refers toany alteration in the state of the body or one of its organs and/ortissues, interrupting or disturbing the performance of organ functionand/or tissue function (e.g., causes organ dysfunction) and/or causing asymptom such as discomfort, dysfunction, distress, or even death to asubject afflicted with the disease.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beincorporated into a pharmaceutical composition administered to a patientwithout causing any significant undesirable biological effects orinteracting in a deleterious manner with any of the other components ofthe composition in which it is contained. When the term“pharmaceutically acceptable” is used to refer to a pharmaceuticalcarrier or excipient, it is implied that the carrier or excipient hasmet the required standards of toxicological and manufacturing testing orthat it is included on the Inactive Ingredient Guide prepared by theU.S. Food and Drug administration.

The term “effective amount” refers to the amount of one or morecompounds that renders a desired treatment outcome. An effective amountmay be comprised within one or more doses, i.e., a single dose ormultiple doses may be required to achieve the desired treatmentendpoint.

The term “therapeutically effective amount” as used herein, refers tothe level or amount of one or more agents needed to treat a condition,or reduce or prevent injury or damage, optionally without causingsignificant negative or adverse side effects.

A “prophylactically effective amount” refers to an amount of an agentsufficient to prevent or reduce severity of a future disease orcondition when administered to a subject who is susceptible and/or whomay develop a disease or condition.

According to the methods of the present invention, the term “subject,”and variants thereof as used herein, includes any subject that has, issuspected of having, or is at risk for having a disease or condition.Suitable subjects (or patients) include mammals, such as laboratoryanimals (e.g., mouse, rat, rabbit, guinea pig), farm animals, anddomestic animals or pets (e.g., cat, dog). Non-human primates and,preferably, human patients, are included. A subject “at risk” may or maynot have detectable disease, and may or may not have displayeddetectable disease prior to the diagnostic or treatment methodsdescribed herein. “At risk” denotes that a subject has one or moreso-called risk factors, which are measurable parameters that correlatewith development of a condition described herein, which are describedherein. A subject having one or more of these risk factors has a higherprobability of developing a condition described herein than a subjectwithout these risk factor(s). One example of such a risk factor is anincrease or decrease in a biomarker of the present invention as comparedto a clinically normal sample.

In certain embodiments, when measuring parameters or other indicators oftreatment, an “increased” or “decreased” amount or level may include a“statistically significant” amount. A result is typically referred to asstatistically significant if it is unlikely to have occurred by chance.The significance level of a test or result relates traditionally to theamount of evidence required to accept that an event is unlikely to havearisen by chance. In certain cases, statistical significance may bedefined as the probability of making a decision to reject the nullhypothesis when the null hypothesis is actually true (a decision knownas a Type I error, or “false positive determination”). This decision isoften made using the p-value: if the p-value is less than thesignificance level, then the null hypothesis is rejected. The smallerthe p-value, the more significant the result. Bayes factors may also beutilized to determine statistical significance (see, e.g., Goodman S.,Ann Intern Med. 130:1005-13, 1999). In some embodiments, an “increased”or “decreased” amount or level is about 1.1×, 1.2×, 1.3×, 1.4×, 1.5×,2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 25×,30×, 40×, or 50× more or less the amount of a predetermined standard, orthe amount of a determined time point relative to a previous or earliertimepoint.

The human subject treated by a composition of the present invention mayshow complete response or partial response. Complete Response (CR), asused herein, unless otherwise indicated, refers to disappearance of allmeasurable and non-measurable symptoms and no appearance of new symptomsin a patient under the treatment. Partial Response (PR), as used herein,unless otherwise indicated, refers to at least one measurable andnon-measurable symptom is significantly reduced, or without appearanceof new symptom in a patient under treatment.

“Phosphate” is distributed in the soft tissue of body in inorganic formor organic form. Non-osseous phosphates comprise less than 20% of thetotal body content. The remainder is stored in the bone matrix.Therefore, the skeleton is the major reservoir of phosphate. The normalrange for serum phosphorus in adults is about 2.5 to about 4.5 mg/dL,and children is higher and varies with age, e.g., the normal range forserum phosphorus in 5-12 years old children is about 2.9 to about 5.7mg/dL (Greenberg et al., The normal range of serum inorganic phosphorusand its utility as a discriminant in the diagnosis of congenitalhypophosphatemia. J Clin Endocrinol Metab 1960; 20:364-379; Burritt etal., Pediatric reference intervals for 19 biologic variables in healthychildren. Mayo Clin Proc 1990; 65:329-336, incorporated by reference inits entirety). When serum phosphate decreases, it is resorbed from bonethrough the activity of PTH and vitamin D. Kidney is the major organregulating phosphate homeostasis. Filtered phosphate is reabsorbedwithin kidney and transported across the renal proximal tubular cellwith the help of NaPi-2A and NaPi-2C.

“Parathyroid Hormone” (PTH) is a regulator of serum calciumconcentration and serum phosphate concentration. Hypocalcemia stimulatesthe production of PTH, which can increase the expression of the proximaltubule 25(OH) vitamin D 1-α-Hydroxylase, an enzyme that synthesizes theactive form of vitamin D, 1,25(OH)₂ vitamin D. 1,25(OH)₂ vitamin Dincreases calcium reabsorption in the renal distal convoluted tubule.The release of calcium from bones into the extracellular fluid is alsostimulated by PTH through increased osteoclastic bone resorption.1,25(OH)₂ vitamin D increases intestinal calcium and phosphateabsorption, and increases phosphate mobilization from bone by increasingosteoclast activity. Both PTH and vitamin D production is influenced byFibroblast growth factor (FGF) 23 in negative feedback loops.

“FGF23” is an endocrine regulator of phosphate homeostasis and wasoriginally identified as the mutated gene in patients with the phosphatewasting disorder “autosomal dominant hypophosphatemic rickets” (ADHR)(Anonymous., “Autosomal Dominant Hypophosphataemic Rickets is Associatedwith Mutations in FGF23,” Nat Genet 26(3):345-348 (2000)). FGF23inhibits reabsorption of phosphate in the renal proximal tubule bydecreasing the abundance of the type II sodium-dependent phosphatetransporters NaPi-2A and NaPi-2C in the apical brush border membrane(Baum et al., “Effect of Fibroblast Growth Factor-23 on PhosphateTransport in Proximal Tubules,” Kidney Int 68(3):1148-1153 (2005);Perwad et al., “Fibroblast Growth Factor 23 Impairs Phosphorus andVitamin D Metabolism In Vivo and Suppresses 25-hydroxyvitaminD-1alpha-hydroxylase Expression In Vitro,” Am J Physiol Renal Physiol293(5): F1577-1583 (2007); Larsson et al., “Transgenic mice expressingfibroblast growth factor 23 under the control of the alpha1(I) collagenpromoter exhibit growth retardation, osteomalacia, and disturbedphosphate homeostasis,” Endocrinology 145(7):3087-3094 (2004)). Thephosphaturic activity of FGF23 is down-regulated by proteolytic cleavageat the 176RXXR179 (SEQ ID NO: 11) motif, where “XX” is defined as “HT”,corresponding to positions 177 and 178, respectively, of the FGF23 aminoacid sequence, producing an inactive N-terminal fragment (Y25 to R179)and a C-terminal fragment (S180 to I251) (Goetz et al., “MolecularInsights into the Klotho-dependent, Endocrine Mode of Action ofFibroblast Growth Factor 19 Subfamily Members,” Mol Cell Biol27(9):3417-3428 (2007)). FGF receptor (FGFR) 1 is the principal mediatorof the phosphaturic action of FGF23 (Liu et al., “FGFR3 and FGFR4 do notMediate Renal Effects of FGF23,” J Am Soc Nephrol 19(12):2342-2350(2008)); Gattineni et al., “FGF23 Decreases Renal NaPi-2a and NaPi-2cExpression and Induces Hypophosphatemia in vivo Predominantly via FGFReceptor 1,” Am J Physiol 297(2): F282-F291 (2009)). In addition,Klotho, a protein first described as an aging suppressor (Kuro-o et al.,“Mutation of the Mouse Klotho Gene Leads to a Syndrome ResemblingAging,” Nature 390(6655):45-51 (1997)), is required as a coreceptor byFGF23 in its target tissue in order to exert its phosphaturic activity(Kurosu et al., “Regulation of Fibroblast Growth Factor-23 Signaling byKlotho,” J Biol Chem 281(10):6120-6123 (2006)); Urakawa et al., “KlothoConverts Canonical FGF Receptor into a Specific Receptor for FGF23,”Nature 444(7120):770-774 (2006); and Goetz R, et al. (IsolatedC-terminal tail of FGF23 alleviates hypophosphatemia by inhibitingFGF23-FGFR-Klotho complex formation. Proc Natl Acad Sci USA. 2009).Klotho constitutively binds the cognate FGFRs of FGF23, and the binaryFGFR-Klotho complexes exhibit enhanced binding affinity for FGF23((Kurosu et al., “Regulation of Fibroblast Growth Factor-23 Signaling byKlotho,” J Biol Chem 281(10):6120-6123 (2006); Urakawa et al., “KlothoConverts Canonical FGF Receptor into a Specific Receptor for FGF23,”Nature 444(7120):770-774 (2006)). In co-immunoprecipitation studies, itwas demonstrated that the mature, full-length form of FGF23 (Y25 toI251) but not the inactive N-terminal fragment of proteolytic cleavage(Y25 to R179) binds to binary FGFR-Klotho complexes (Goetz et al.,“Molecular Insights into the Klotho-dependent, Endocrine Mode of Actionof Fibroblast Growth Factor 19 Subfamily Members,” Mol Cell Biol27(9):3417-3428 (2007)). High levels of FGF23 signaling in vitro occurwhen KL and FGFR1c are co-expressed, and this activity can be blocked byanti-FGF23 antibodies that disrupt FGFR-FGF23-KL associations (UrakawaI, et al. Klotho converts canonical FGF receptor into a specificreceptor for FGF23. Nature 2006; 444:770-774; Aono Y, et al. TherapeuticEffects of Anti-FGF23 Antibodies in HypophosphatemicRickets/Osteomalacia. J Bone Miner Res. 2009).

FGF23 has the function of reducing phosphate reabsorption in proximaltubule, by reducing expression of the renal vitamin D1-α-hydroxylase andincreasing expression of the catabolic 25(OH) D-24-hydroxylase, thusdecreasing circulating 1,25(OH)₂D concentrations. Overexpression ofFGF23 in vivo leads to hypophosphatemia, rickets/osteomalacia, similarto patients with ADHR, X-linked hypophosphatemia (XLH), andtumor-induced osteomalacia (TIO) (Larsson T, et al. Transgenic miceexpressing fibroblast growth factor 23 under the control of thealpha1(I) collagen promoter exhibit growth retardation, osteomalacia,and disturbed phosphate homeostasis. Endocrinology 2004; 145:3087-3094;Shimada T, et al. FGF-23 transgenic mice demonstrate hypophosphatemicrickets with reduced expression of sodium phosphate cotransporter typeIIa. Biochem Biophys Res Commun 2004; 314:409-414). On the other hand,1,25(OH)₂D stimulates FGF23 promoter activity in vitro and production invivo (Kolek O I, et al. 1alpha, 25-Dihydroxyvitamin D3 upregulates FGF23gene expression in bone: the final link in arenal-gastrointestinal-skeletal axis that controls phosphate transport.Am J Physiol Gastrointest Liver Physiol 2005; 289: G1036-G1042; Liu S,et al. Novel regulators of Fgf23 expression and mineralization in Hypbone. Mol Endocrinol 2009; 23:1505-1508; Shimada T, et al. FGF-23 is apotent regulator of vitamin D metabolism and phosphate homeostasis. JBone Miner Res 2004; 19:429-435), suggesting there is a negativefeedback process between kidney and bone.

The “Klotho” protein is a 130-kDa single-pass transmembrane proteinexpressed predominantly in the kidney (Matsumara et al., “Identificationof the human klotho gene and its two transcripts encoding membrane andsecreted klotho protein,” Biochem Biophys Res Commun 242(3):626-630(1998), which is hereby incorporated by reference in its entirety). Inaddition to the membrane-bound isoform of Klotho, alternative splicingand proteolytic cleavage give rise to two soluble isoforms of Klothofound in the circulation (Imura et al., “Secreted Klotho protein in seraand CSF: implication for post-translational cleavage in release ofKlotho protein from cell membrane,” FEBS Lett 565(1-3): 143-147 (2004);Kurosu et al., “Suppression of aging in mice by the hormone Klotho,”Science 309(5742):1829-1833 (2005); Matsumura et al., “Identification ofthe human klotho gene and its two transcripts encoding membrane andsecreted klotho protein,” Biochem Biophys Res Commun 242(3):626-630(1998); Shiraki-Lida et al., “Structure of the mouse klotho gene and itstwo transcripts encoding membrane and secreted protein,” FEBS Lett424(1-2): 6-10 (1998), which are hereby incorporated by reference intheir entirety). Observations suggest that the Klotho gene functions asan aging suppressor gene.

FGFR1, transcript variant 1 is a member of the fibroblast growth factorreceptor (FGFR) family that is highly conserved between members andthroughout evolution. A full-length representative protein consists ofan extracellular region, composed of three immunoglobulin-like domains,a single hydrophobic membrane-spanning segment, and a cytoplasmictyrosine kinase domain. The extracellular portion of the proteininteracts with fibroblast growth factors, setting in motion a cascade ofdownstream signals, ultimately influencing mitogenesis anddifferentiation. This particular family member binds both acidic andbasic fibroblast growth factors and is involved in limb induction.Mutations in this gene have been associated with Pfeiffer syndrome,Jackson-Weiss syndrome, Antley-Bixler syndrome, osteoglophonicdysplasia, and autosomal dominant Kallmann syndrome. See Itoh et al.,“The Complete Amino Acid Sequence of the Shorter Form of Human BasicFibroblast Growth Factor Receptor Deduced from its cDNA,” BiochemBiophys Res Commun 169(2): 680-685 (1990); Dode et al., “KallmannSyndrome: Fibroblast Growth Factor Signaling Insufficiency?” J Mol Med82(11):725-34 (2004); Coumoul et al., “Roles of FGF Receptors inMammalian Development and Congenital Diseases,” Birth Defects Res CEmbryo Today 69(4):286-304 (2003), which are hereby incorporated byreference in their entirety. Alternatively, spliced variants whichencode different protein isoforms have been described; however, not allvariants have been fully characterized.

“Human FGF23” has the sequence of SEQ ID NO: 12, and the mouse FGF23 hasthe sequence of SEQ ID NO: 13. FGF23 is inactivated throughintracellular proteolysis at the subtilisin-like proprotein convertase(SPC) site R176HTR179/S180 (SEQ ID NO: 14). Some FGF23 mutants, such asR176Q, R179Q, and R179W, destroy this site and stabilize the full-lengthactive form of the protein, which leads to ADHR symptoms. Human Klothohas the sequence of SEQ ID NO: 15.

In mammals, FGFs mediate their action via a set of four FGF receptors,FGFR1-4, that in turn are expressed in multiple spliced variants, e.g.,FGFR1c, FGFR2c, FGFR3c and FGFR4. Each FGF receptor contains anintracellular tyrosine kinase domain that is activated upon ligandbinding, leading to downstream signaling pathways involving MAPKs(Erk1/2), RAF1, AKT1 and STATs. (Kharitonenkov et al., (2008) BioDrugs22:37-44). Several reports suggested that the “c”-reporter splicevariants of FGFR1-3 exhibit specific affinity to β-Klotho and could actas endogenous receptor for FGF21 (Kurosu et al., (2007) J. Biol. Chem.282:26687-26695); Ogawa et al., (2007) Proc. Natl. Acad. Sci. USA104:7432-7437); Kharitonenkov et al., (2008) J. Cell Physiol. 215:1-7).In the liver, which abundantly expresses both β-Klotho and FGFR4, FGF21does not induce phosphorylation of MAPK albeit the strong binding ofFGF21 to the β-Klotho-FGFR4 complex. In 3T3-L1 cells and white adiposetissue, FGFR1 is by far the most abundant receptor, and it is thereforemost likely that FGF21's main functional receptors in this tissue arethe β-Klotho-FGFR1c complexes. Alternative splicing of FGFR geneproduces multiple FGFR isoforms.

“Hyperphosphatemia” is an electrolyte disturbance in which there is anabnormally elevated level of phosphate in the blood. Often, calciumlevels are lowered (hypocalcemia) due to precipitation of phosphate withthe calcium in tissues. Average phosphorus levels should be betweenabout 0.81 mmol/L to about 1.45 mmol/L. Signs and symptoms include, butare not limited to, ectopic calcification, secondaryhyperparathyroidism, and renal osteodystrophy. Hyperphosphatemia may becaused by impaired renal phosphate excretion and/or massiveextracellular fluid phosphate loads. Impaired renal phosphate excretionmay be due to hypoparathyroidism (e.g., developmental, autoimmune, aftersurgery or radiation, or activating mutations of calcium-sensingreceptor), parathyroid suppression (e.g., Parathyroid-independenthypercalcemia, vitamin D or vitamin A intoxication, sarcoidosis, othergranulomatous diseases, immobilization, osteolytic metastases,milk-alkali syndrome, or severe hypermagnesemia or hypomagnesemia),pseudohyporarathyroidism, acromegaly, tumoral calcinosis, or heparintherapy. Massive extracellular fluid phosphate loads may be due to rapidadministration of exogenous phosphate, extensive cellular injury ornecrosis (e.g., crush injuries, rhadbomyolysis, hyperthermia, fulminanthepatitis, cytotoxic therapy, or severe hemolytic anemia), ortrans-cellular phosphate shifts.

In contrast, hypophosphatemia refers to serum phosphate concentrationbelow the normal range of 2.2 to 4.9 mg/dl (Dwyer et al., “Severehypophosphatemia in postoperative patients,” Nutr Clin Pract7(6):279-283 (1992); Alon et al., “Calcimimetics as an adjuvanttreatment for familial hypophosphatemic rickets,” Clin J Am Soc Nephrol3(3):658-664 (2008), which are hereby incorporated by reference in theirentirety).

Hypophosphatemia may be due to renal phosphate wasting (such as,autosomal dominant hypophosphatemic rickets (ADHR), X-linkedhypophosphatemia (XLH), autosomal recessive hypophosphatemic rickets(ARHR), fibrous dysplasia (FD), McCune-Albright syndrome complicated byfibrous dysplasia (MAS/FD), Jansen's metaphyseal chondrodysplasia(Jansen's Syndrome), autosomal dominant polycystic kidney disease(ADPKD), tumor-induced osteomalacia (TIO), and chronic metabolicacidosis), other inherited or acquired renal phosphate wastingdisorders, alcoholic and diabetic ketoacidosis, acute asthma, chronicobstructive pulmonary disease (COPD), drug treatment of COPD, sepsis,recovery from organ (in particular, kidney) transplantation, parenteraliron administration, salicylate intoxication, severe trauma, chronictreatment with sucralfate and/or antacids, mechanical ventilation,eating disorder (such as, anorexia nervosa and bulimia nervosa), or therefeeding syndrome.

“Renal phosphate wasting” refers to an inherited or acquired conditionin which renal tubular reabsorption of phosphate is impaired. It occursin approximately 50% of patients with McCune-Albright syndrome (MAS) andfibrous dysplasia of bone (FD). It is reported that serum levels ofFGF23 were increased in FD/MAS patients compared with normal age-matchedcontrols and significantly higher in FD/MAS patients with renalphosphate wasting compared with those without, and correlated withdisease burden bone turnover markets commonly used to assess diseaseactivity (Riminucci et al., FGF-23 in fibrous dysplasia of bone and itsrelationship to renal phosphate wasting, J. Clin. Invest. 112:683-692(2003)).

Methods

According to the present invention, it provides methods of usinganti-FGF23 ligands, especially methods of determining the dosing regimenof anti-FGF23 ligands, based on one or more PD parameters of theanti-FGF23 ligands in subjects receiving treatments. In someembodiments, one or more anti-FGF23 ligands are used for the treatmentof conditions associated with FGF23, e.g., higher than normal level ofFGF23. For example, in some embodiments, anti-FGF23 ligands are used totreat Autosomal dominant hypophosphatemic rickets/osteomalachia (ADHR),X-linked hypophosphatemia (XLH), autosomal recessive hypophosphatemicrickets (ARHR), fibrous dysplasia (FD), McCune-Albright syndromecomplicated by fibrous dysplasia (MAS/FD), Jansen's metaphysealchondrodysplasia (Jansen's Syndrome), autosomal dominant polycystickidney disease (ADPKD), tumor-induced osteomalacia (TIO), chronicmetabolic acidosis and ectopic calcification.

In some other embodiments, the anti-FGF23 ligands are used to treatdisorders or conditions associated with hypophosphatemia or renalphosphate wasting.

According to the present invention, anti-FGF23 ligands can include anyactive ingredient or agent that can modulate, e.g., down regulate theactivity of FGF23 and/or one or more components in the FGF23 signalingpathway thereby down regulate the activity of FGF23. In someembodiments, the active agent is a small molecule. In some embodiments,the active agent is a polypeptide, such as an antibody. In someembodiments, the active agent is a polynucleotide, such as siRNA. Asused herein, the term activity refers to the activity of a given targetat the genomic DNA level, transcriptional level, post-transcriptionallevel, translational level, post-translational level, including but notlimited to, gene copy number, mRNA transcription rate, mRNA abundance,mRNA stability, protein translation rate, protein stability, proteinmodification, protein activity, protein complex activity, etc.

In some embodiments, the active agent modulates the activity of FGF23.In some embodiments, the active agent modulates gene copy number of acomponent in the FGF23 signaling pathway. In some embodiments, theactive agent can increase or decrease the gene copy number by 0.5×,1.0×, 1.5×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 100×, 1000×, 10000× ormore when compared to the gene copy number before treatment.

In some embodiments, the active agent modulates the mRNA abundance of acomponent in the FGF23 signaling pathway. In some embodiments, theactive agent can increase or decrease the mRNA abundance by 0.5×, 1.0×,1.5×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 100×, 1000×, 10000× or morewhen compared to the mRNA abundance before treatment.

In some embodiments, the active agent modulates the protein level of acomponent in the FGF23 signaling pathway. In some embodiments, theactive agent can increase or decrease the protein level by 0.5×, 1.0×,1.5×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 100×, 1000×, 10000× or morewhen compared to the protein level before treatment.

In some embodiments, the active agent modulates the mRNA and/or proteinactivity or stability of a component in the FGF23 signaling pathway. Insome embodiments, the active agent can increase or decrease the activityor stability when compared to the stability before treatment.

In some embodiments, the active agent modulates the enzymatic activityof a component in the FGF23 signaling pathway. In some embodiments, theactive agent can increase or decrease the enzymatic activity whencompared to the stability before treatment.

The activity of a component in the FGF23 signaling pathway can bedetermined by any suitable methods known to one skilled in the art. Insome embodiments, a biological sample is taken from a subject andanalyzed. In some embodiments, the biological sample is then assayed foractivity of a component in the FGF23 signaling pathway, such as geneamplification number, RNA, mRNA, cDNA, cRNA, protein, etc.

In some embodiments, mRNA from a biological sample is directly used indetermining the level of activity. In some embodiments, the level isdetermined by hybridization. In some embodiments, the RNA is transformedinto cDNA (complementary DNA) copy using methods known in the art. Insome particular embodiments, the cDNA is labeled with a fluorescentlabel or other detectable label. The cDNA is then hybridized to asubstrate containing a plurality of probes of interest. A probe ofinterest typically hybridizes under stringent hybridization conditionsto at least one DNA sequence of a gene signature. In certainembodiments, the plurality of probes are capable of hybridizing to thesequences derived from the gene biomarkers under the hybridizationconditions. In some embodiments, the conditions comprise using 6×SSC(0.9 M NaCl, 0.09 M sodium citrate, pH 7.4) at 65° C. The probes maycomprise nucleic acids. The term “nucleic acid” encompasses knownnucleotide analogs or modified backbone residues or linkages, which aresynthetic, naturally occurring, and non-naturally occurring, which havesimilar binding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,peptide-nucleic acids (PNAs). Methods for detecting can include but arenot limited to RT-PCR, northern blot analyses, gene expression analyses,microarray analyses, gene expression chip analyses, hybridizationtechniques (including FISH), expression beadchip arrays, andchromatography as well as any other techniques known in the art. Methodsfor detecting DNA can include but are not limited to PCR, real-time PCR,digital PCR, hybridization (including FISH), microarray analyses, SNPdetection assays, SNP genotyping assays and chromatography as well asany other techniques known in the art.

In some embodiments, the protein expression level is used in determiningthe level of activity. The protein expression level of a component inthe FGF23 signaling pathway can be determined by any suitable methodsknown to one skilled in the art. Any suitable methods of proteindetection, quantization and comparison can be used, such as thosedescribed in Tschesche (Methods in Protein Biochemistry, ISBN Walter deGruyter, 2011, ISBN 3110252368, 9783110252361), Goluch et al.(Chip-based detection of protein cancer markers, ProQuest, 2007, ISBN0549463453, 9780549463450), Speicher (Proteome Analysis: Interpretingthe Genome, Elsevier, 2004, ISBN 0080515304, 9780080515304), Albala etal. (Protein Arrays, Biochips and Proteomics, CRC Press, 2003, ISBN0203911121, 9780203911129), Walker (The Protein Protocols Handbook,Springer, 2002, ISBN 0896039404, 9780896039407), Fung (Protein Arrays:Methods and Protocols, Springer, 2004, ISBN 1592597599, 9781592597598),and Bienvenut (Acceleration and Improvement of Protein Identification byMass Spectrometry, Springer, 2005, ISBN 1402033184, 9781402033186), eachof which is incorporated by reference in its entirety for all purposes.In some embodiments, the protein expression level of biomarkers aredetected and measured by immunohistochemistry (IHC), western blot,protein immunostaining, protein immunoprecipitation,immunoeletrophoresis, immunoblotting, BCA assay, spectrophotometry, massspectrometry or enzyme assay, or combinations thereof. For additionalmethods related to detection, quantitation and comparison of biomarkerlevels, see, e.g., Current Protocols in Molecular Biology, Ed. Ausubel,Frederick M. (2010); Current Protocols in Protein Science Last, Ed.Coligan, John E., et al. (2010); Current Protocols in Nucleic AcidChemistry, Ed. Egli, Martin (2010); Current Protocols in Bioinformatics,Ed. Baxevanis, Andreas D. (2010); and Molecular Cloning: A LaboratoryManual, Third Edition, Sambrook, Joseph (2001), all of which areincorporated herein by reference in their entirety.

In some embodiments, an antibody directed to a component in the FGF23signaling pathway can be used as an active agent. In some embodiments,an antibody directed to a component in the FGF23 signaling pathway thatpositively controls FGF23, or an antibody directed to a component in theFGF23 signaling pathway that is positively controlled by FGF23 can beused as an active agent.

In some embodiments, the active agent can directly decrease the FGF23activity in a human subject. In some embodiments, the active agent canmodulate the activity of one or more upstream or downstream componentsin the signaling pathway thereby decreasing the FGF23 activity in ahuman subject indirectly. In some embodiments, the active agent candecrease the activity of one or more upstream components that positivelyregulate FGF23, or increase the activity of one or more upstreamcomponents in the signaling pathway that negatively regulate FGF23. Insome embodiments, the active agent can decrease the activity of one ormore downstream components that are positively regulated by FGF23, orincrease the activity of one or more downstream components in thesignaling pathway that are negatively regulated by FGF23.

Without wishing to be bound by any particular theory, the agents of thepresent invention can act through one or more mechanisms. Suchmechanisms include, but are not limited to: (1) inhibition of FGF23activity; (2) inhibition of FGF23-Klotho-FGF receptor complex activity;(3) increasing phosphate reabsorption in proximal tubule; (4) increasingactivity of the renal vitamin D1-α-hydroxylase; (5) decreasing theactivity of catabolic 25(OH) D-24-hydroxylase; and/or (6) increasing1,25(OH)₂D concentrations.

The active agents can be chemical compounds or compositions, biologicalmolecules, or combinations thereof. In some embodiments, the activeagents are small molecules. As used herein, the term “small molecule”refers to a molecule having a molecular weight of less than 500 MW,wherein the drug is a non-peptidyl or peptide agent. In someembodiments, the active agents are antibodies. In some embodiments, theactive agents are antibodies. In some embodiments, the active agents arepolynucleotides, such as siRNA.

In some embodiments, the active agents contain one or more antibodiesthat can reduce, inhibit or delay the activity of a component in theFGF23 signaling pathway that positively regulates FGF23, or ispositively regulated by FGF23. In some embodiments, the component can bea member of the FGF23-Klotho-FGF receptor complex. In some embodiments,the component is FGF23. In some embodiments, the agent is a monoclonalantibody. In some embodiments, the antibody is an antibody describedherein. In some embodiments, an anti-FGF23 ligand is an active agentcomprising one or more anti-FGF23 CDRs, such as those of SEQ ID NOs.1-6.

In some embodiments, the active agent is an antibody against human FGF23or a functional fragment thereof, comprising a heavy chain variableregion having any one of complementarity determining region (CDR) 1shown by the amino acid sequence of SEQ ID NO: 1, CDR2 shown by theamino acid sequence of SEQ ID NO: 2 and CDR3 shown by the amino acidsequence of SEQ ID NO: 3, or a heavy chain variable region having atleast two or all of the three heavy chain CDRs.

In some embodiments, the antibody against human FGF23 or a functionalfragment thereof, comprises a light chain variable region having any oneof CDR1 shown by the amino acid sequence of SEQ ID NO: 4, CDR2 shown bythe amino acid sequence of SEQ ID NO: 5 and CDR3 shown by the amino acidsequence of SEQ ID NO: 6, or a light chain variable region having atleast two or all of the three light chain CDRs.

In some embodiments, the FGF23 antibody contains CDRs with amino acidsequence of SEQ ID NO: 1, 2, 3, 4, 5, and/or 6. In some embodiments, theFGF23 antibody comprises the heavy chain variable region comprising SEQID NO: 16 and the light chain variable region comprising SEQ ID NO: 17.In some embodiments, the FGF23 antibody is KRN23 having the heavy chainof SEQ ID NO: 7 and the light chain of SEQ ID NO: 8. In someembodiments, the FGF23 antibody is C10 having the heavy chain of SEQ IDNO: 9 and the light chain of SEQ ID NO: 10.

The CDR sequence of the antibody of the present invention is notspecifically limited. In some embodiments, the antibody of the presentinvention is an antibody comprising any one or more CDRs, morepreferably three CDRs of the heavy chain, and even more preferably sixCDRs of the CDR sequences represented by SEQ ID NOs: 1 through 6. Theamino acid sequence other than the CDR is not specifically limited. Insome other embodiments, anti-FGF23 antibodies include so called CDRtransplantation antibodies, wherein the amino acid sequence other thanthe CDR is derived from other antibodies, and particularly antibodies inother species. Among these, a humanized antibody or human antibody,wherein the amino acid sequence other than the CDR is derived fromhuman, is preferred. An addition, deletion, substitution and/orinsertion of 1 amino acid residue or more can be introduced into the FRaccording to need. A publicly known method can be applied as the methodfor producing a humanized antibody or human antibody.

In some embodiments, the antibody against FGF23 is anyone of thosedescribed in U.S. Pat. Nos. 7,314,618, 7,223,563, 7,745,406, 7,947,810,7,223,563, 7,745,406 or 7,947,810, or in U.S. Patent ApplicationPublication Nos. 20120064544 or 20110182913, each of which isincorporated by reference in its entirety for all purposes.

In some embodiments, the active agents are siRNA. For example, antisenseRNA, ribozyme, dsRNAi, RNA interference (RNAi) technologies can be usedin the present invention to target RNA transcripts of one or morecomponent in the FGF23 signaling pathway. Antisense RNA technologyinvolves expressing in, or introducing into, a cell an RNA molecule (orRNA derivative) that is complementary to, or antisense to, sequencesfound in a particular mRNA in a cell. By associating with the mRNA, theantisense RNA can inhibit translation of the encoded gene product.

RNA interference (RNAi) is the process of sequence-specific,post-transcriptional gene silencing or transcriptional gene silencing inanimals and plants, initiated by double-stranded RNA (dsRNA) that ishomologous in sequence to the silenced gene. The RNAi technique isdiscussed, for example, in Elibashir, et al., Methods Enzymol. 26:199(2002); McManus & Sharp, Nature Rev. Genetics 3:737 (2002); PCTapplication WO 01/75164; Martinez et al., Cell 110:563 (2002); Elbashiret al., supra; Lagos-Quintana et al., Curr. Biol. 12:735 (2002); Tuschlet al., Nature Biotechnol. 20:446 (2002); Tuschl, Chembiochem. 2:239(2001); Harborth et al., J. Cell Sci. 114:4557 (2001); et al., EMBO J.20:6877 (2001); Lagos-Quintana et al., Science 294:8538 (2001);Hutvagner et al., loc cit, 834; Elbashir et al., Nature 411:494 (2001).

The term “dsRNA” or “dsRNA molecule” or “double-strand RNA effectormolecule” refers to an at least partially double-strand ribonucleic acidmolecule containing a region of at least about 19 or more nucleotidesthat are in a double-strand conformation. The double-stranded RNAeffector molecule may be a duplex double-stranded RNA formed from twoseparate RNA strands or it may be a single RNA strand with regions ofself-complementarity capable of assuming an at least partiallydouble-stranded hairpin conformation (i.e., a hairpin dsRNA or stem-loopdsRNA). In various embodiments, the dsRNA consists entirely ofribonucleotides or consists of a mixture of ribonucleotides anddeoxynucleotides, such as RNA/DNA hybrids. The dsRNA may be a singlemolecule with regions of self-complementarity such that nucleotides inone segment of the molecule base pair with nucleotides in anothersegment of the molecule. In one aspect, the regions ofself-complementarity are linked by a region of at least about 3-4nucleotides, or about 5, 6, 7, 9 to 15 nucleotides or more, which lackscomplementarity to another part of the molecule and thus remainssingle-stranded (i.e., the “loop region”). Such a molecule will assume apartially double-stranded stem-loop structure, optionally, with shortsingle stranded 5′ and/or 3′ ends. In one aspect the regions ofself-complementarity of the hairpin dsRNA or the double-stranded regionof a duplex dsRNA will comprise an Effector Sequence and an EffectorComplement (e.g., linked by a single-stranded loop region in a hairpindsRNA). The Effector Sequence or Effector Strand is that strand of thedouble-stranded region or duplex which is incorporated in or associateswith RISC. In one aspect the double-stranded RNA effector molecule willcomprise an at least 19 contiguous nucleotide effector sequence,preferably 19 to 29, 19 to 27, or 19 to 21 nucleotides, which is areverse complement to the RNA of a target gene, or an opposite strandreplication intermediate, or the anti-genomic plus strand or non-mRNAplus strand sequences of the target gene.

In some embodiments, the dsRNA effector molecule of the invention is a“hairpin dsRNA”, a “dsRNA hairpin”, “short-hairpin RNA” or “shRNA”,i.e., an RNA molecule of less than approximately 400 to 500 nucleotides(nt), or less than 100 to 200 nt, in which at least one stretch of atleast 15 to 100 nucleotides (e.g., 17 to 50 nt, 19 to 29 nt) is basedpaired with a complementary sequence located on the same RNA molecule(single RNA strand), and where said sequence and complementary sequenceare separated by an unpaired region of at least about 4 to 7 nucleotides(or about 9 to about 15 nt, about 15 to about 100 nt, about 100 to about1000 nt) which forms a single-stranded loop above the stem structurecreated by the two regions of base complementarity. The shRNA moleculescomprise at least one stem-loop structure comprising a double-strandedstem region of about 17 to about 100 bp; about 17 to about 50 bp; about40 to about 100 bp; about 18 to about 40 bp; or from about 19 to about29 bp; homologous and complementary to a target sequence to beinhibited; and an unpaired loop region of at least about 4 to 7nucleotides, or about 9 to about 15 nucleotides, about 15 to about 100nt, about 100 to about 1000 nt, which forms a single-stranded loop abovethe stem structure created by the two regions of base complementarity.It will be recognized, however, that it is not strictly necessary toinclude a “loop region” or “loop sequence” because an RNA moleculecomprising a sequence followed immediately by its reverse complementwill tend to assume a stem-loop conformation even when not separated byan irrelevant “stuffer” sequence.

In yet other embodiments, anti-FGF23 ligands used in the presentinvention can be provided in pharmaceutical compositions comprising oneor more of the active agents used in the present invention and avehicle, such as an artificial membrane vesicle (including a liposome,lipid micelle and the like), microparticle or microcapsule.

Compositions intended for oral use may be prepared in either solid orfluid unit dosage forms. Fluid unit dosage form can be preparedaccording to procedures known in the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents selected from the group consisting of sweetening agents,flavouring agents, colouring agents and preserving agents in order toprovide pharmaceutically elegant and palatable preparations. An elixiris prepared by using a hydroalcoholic (e.g., ethanol) vehicle withsuitable sweeteners such as sugar and saccharin, together with anaromatic flavoring agent. Suspensions can be prepared with an aqueousvehicle with the aid of a suspending agent such as acacia, tragacanth,methylcellulose and the like.

Solid formulations such as tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients that aresuitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate: granulating anddisintegrating agents for example, corn starch, or alginic acid: bindingagents, for example starch, gelatin or acacia, and lubricating agents,for example magnesium stearate, stearic acid or talc and otherconventional ingredients such as dicalcium phosphate, magnesium aluminumsilicate, calcium sulfate, starch, lactose, methylcellulose, andfunctionally similar materials. The tablets may be uncoated or they maybe coated by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil. Softgelatin capsules are prepared by machine encapsulation of a slurry ofthe compound with an acceptable vegetable oil, light liquid petrolatumor other inert oil.

Aqueous suspensions contain active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxylmethylcellulose, methyl cellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia:dispersing or wetting agents may be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample hepta-decaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl-p-hydroxy benzoate, one or more colouringagents, one or more flavouring agents or one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil, for example peanut oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavouring agents may be added to provide palatable oralpreparations. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavouring and colouringagents, may also be present.

Pharmaceutical compositions of the invention may also be in the form ofoil-in-water emulsions. The oil phase may be a vegetable oil, forexample olive oil or peanut oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to known art using those suitable dispersing orwetting agents and suspending agents that have been mentioned above. Thesterile injectable preparation may also be a sterile injectable solutionor a suspension in a non-toxic parentally acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables. Adjuvants such as local anesthetics,preservatives and buffering agents can also be included in theinjectable solution or suspension.

In some embodiments, the delivery systems suitable include time-release,delayed release, sustained release, or controlled release deliverysystems. In some embodiments, a composition of the present invention canbe delivered in a controlled release system, such as sustained-releasematrices. Non-limiting examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., 1981, J. Biomed. Mater. Res., 15:167-277 andLanger, 1982, Chem. Tech., 12:98-105), or poly(vinylalcohol)],polylactides (U.S. Pat. No. 3,773,919; EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983,Biopolymers, 22:547-556), non-degradable ethylene-vinyl acetate (Langeret al., supra), degradable lactic acid-glycolic acid copolymers such asthe LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). In some embodiments, thecomposition may be administered using intravenous infusion, animplantable osmotic pump, a transdermal patch, liposomes, or other modesof administration. In one embodiment, a pump may be used (see Langer,supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald etal., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574(1989). In another embodiment, polymeric materials can be used. In yetanother embodiment, a controlled release system can be placed inproximity to the therapeutic target, for example liver, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984).Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990). In some embodiments, the composition maybe administered through subcutaneous injection.

In some embodiments, the release of the composition occurs in bursts.Examples of systems in which release occurs in bursts includes, e.g.,systems in which the composition is entrapped in liposomes which areencapsulated in a polymer matrix, the liposomes being sensitive tospecific stimuli, e.g., temperature, pH, light or a degrading enzyme andsystems in which the composition is encapsulated by an ionically-coatedmicrocapsule with a microcapsule core degrading enzyme.

In some embodiments, the release of the composition isgradual/continuous.

Examples of systems in which release of the inhibitor is gradual andcontinuous include, e.g., erosional systems in which the composition iscontained in a form within a matrix and effusional systems in which thecomposition is released at a controlled rate, e.g., through a polymer.Such sustained release systems can be e.g., in the form of pellets, orcapsules.

Other embodiments of the compositions administered according to theinvention incorporate particulate forms, protective coatings, proteaseinhibitors or permeation enhancers for various routes of administration,such as parenteral, pulmonary, nasal and oral.

The pharmaceutical compositions of the present invention can be usedalone or in combination with other pharmaceutical compositions. Thepharmaceutical compositions of the present invention may beadministered, together or separately, in the form of suppositories forrectal administration of the drug. These compositions can be prepared bymixing the drug with a suitable non-irritating excipient which is solidat ordinary temperatures but liquid at the rectal temperature and willtherefore melt in the rectum to release the drug. Such materials includecocoa butter and polyethylene glycols. In some embodiments, the apharmaceutical composition of the present invention may be administeredto a subject together with an addition pharmaceutical compositionsuitable for treating disorders associated with abnormal phosphorusstatus, FGF23 signaling, or bone formation.

Other pharmaceutical compositions and methods of preparingpharmaceutical compositions are known in the art and are described, forexample, in “Remington: The Science and Practice of Pharmacy” (formerly“Remingtons Pharmaceutical Sciences”); Gennaro, A., Lippincott, Williams& Wilkins, Philadelphia, Pa. (2000).

In some embodiments, administration of a composition of the presentinvention may be carried out orally, parenterally, subcutaneously,intravenously, intramuscularly, intraperitoneally, by intranasalinstillation, by implantation, by intracavitary or intravesicalinstillation, intraocularly, intraarterially, intralesionally,transdermally, or by application to mucous membranes. The inhibitor maybe administered with a pharmaceutically-acceptable carrier.

The dosage to be administered is not subject to defined limits, but itwill usually be an effective amount. It will usually be the equivalent,on a molar basis of the pharmacologically active free form produced froma dosage formulation upon the metabolic release of the active free drugto achieve its desired pharmacological and physiological effects. Thecompositions may be formulated in a unit dosage form. The term “unitdosage form” refers to physically discrete units suitable as unitarydosages for human subjects and other mammals, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect, in association with a suitablepharmaceutical excipient.

According to the present invention, the dosing regimen of an anti-FGF23ligand can be determined based on one or more PD parameters of theanti-FGF23 ligand. In some embodiments, the dosing regimen of ananti-FGF23 ligand includes, without any limitation, the amount per dose,frequency of dosing, e.g., per day, week, or month, total amount perdosing cycle, dosing interval, dosing variation, pattern or modificationper dosing cycle, maximum accumulated dosing, or warm up dosing, or anycombination thereof. In some other embodiments, the dosing regimen of ananti-FGF23 ligand includes frequency of dosing, e.g., per month.

In yet some other embodiments, the dosing regimen includes apre-determined or fixed amount per dose in combination with a frequencyof such dose. For example, the dosing regimen of an anti-FGF23 antibodyincludes a fixed amount of anti-FGF23 antibody per dose in combinationwith the frequency of such dose of antibody being administered to asubject. In some embodiments, the fixed amount of anti-FGF23 antibodyper dose includes without any limitation about 0.001 mg/kg of bodyweight, about 0.002 mg/kg of body weight, about 0.003 mg/kg of bodyweight, about 0.004 mg/kg of body weight, about 0.005 mg/kg of bodyweight, about 0.006 mg/kg of body weight, about 0.007 mg/kg of bodyweight, about 0.008 mg/kg of body weight, about 0.009 mg/kg of bodyweight, about 0.01 mg/kg of body weight, about 0.02 mg/kg of bodyweight, about 0.03 mg/kg of body weight, about 0.04 mg/kg of bodyweight, about 0.05 mg/kg of body weight, about 0.06 mg/kg of bodyweight, about 0.07 mg/kg of body weight, about 0.08 mg/kg of bodyweight, about 0.09 mg/kg of body weight, about 0.1 mg/kg of body weight,about 0.2 mg/kg of body weight, about 0.3 mg/kg of body weight, about0.4 mg/kg of body weight, about 0.5 mg/kg of body weight, about 0.6mg/kg of body weight, about 0.7 mg/kg of body weight, about 0.8 mg/kg ofbody weight, about 0.9 mg/kg of body weight, about 1 mg/kg of bodyweight, about 2 mg/kg of body weight, about 3 mg/kg of body weight,about 4 mg/kg of body weight, about 5 mg/kg of body weight, about 6mg/kg of body weight, about 7 mg/kg of body weight, about 8 mg/kg ofbody weight, about 9 mg/kg of body weight, about 10 mg/kg of bodyweight, about 15 mg/kg of body weight, about 20 mg/kg of body weight,about 30 mg/kg of body weight, about 35 mg/kg of body weight, about 40mg/kg of body weight, about 45 mg/kg of body weight, about 50 mg/kg ofbody weight, about 60 mg/kg of body weight, about 70 mg/kg of bodyweight, about 80 mg/kg of body weight, about 90 mg/kg of body weight,about 100 mg/kg of body weight, about 200 mg/kg of body weight, about300 mg/kg of body weight, about 400 mg/kg of body weight, about 500mg/kg of body weight or more.

In some other embodiments, the fixed amount of anti-FGF23 antibody perdose includes without any limitation about 0.05 mg/kg, 0.1 mg/kg, 0.3mg/kg, 0.6 mg/kg, or 1.0 mg/kg or 2.0 mg/kg e. g., administered SC.

According to the present invention, the dosing regimen of an anti-FGF23ligand can be determined based on one or more PD parameters of theanti-FGF23 ligand. In some embodiments, the dosing regimen of ananti-FGF23 ligand is determined based on one or more PD parameters ofthe anti-FGF23 ligand in a population of subjects treated with theanti-FGF23 ligand. In some other embodiments, the size of the populationincludes at least 20, 30, 40, 50, or more subjects. In some otherembodiments, the size of the population provides statisticalsignificance for the analysis of one or more PD parameters. In someother embodiments, the dosing regimen of an anti-FGF23 ligand isdetermined based on one or more PD parameters of the anti-FGF23 ligandin the single subject treated with the anti-FGF23 ligand. For example,one or more PD parameters can be measured in a subject treated with ananti-FGF23 ligand for a period of time, then the dosing regimen of thesubject can be modified based on the subject's PD parameters measured.In other words, one or more PD parameters can be used to “tailor” thedosing regimen of a subject under treatment with an anti-FGF23 ligand.

In some other embodiments, the dosing regimen of an anti-FGF23 ligand isdetermined based on a pre-determined level or activity of one or more PDparameters. For example, such pre-determined level or activity can beobtained via treating a population of patients with an anti-FGF23 ligandand determining the desired or standard level or activity for one ormore PD parameters.

In general, pharmacodynamics (PD) is the study of the biochemical andphysiological effects of drugs on the body or on microorganisms orparasites within or on the body and the mechanisms of drug action andthe relationship between drug concentration and effect. Pharmacodynamicsis often summarized as the study of what a drug does to the body. Theeffect of an active agent of the present invention can be determined bymonitoring the changes of one or more PD parameters associated with thetreatment, so an effective dosage regimen of the active agent can bedetermined. In some embodiments, the dosing regimen is determinedwithout any input from one or more PK parameters or without givingconsideration of one or more PK parameters. In some other embodiments,the dosing regimen is modified even though one or more PK parametersindicate that the dosing regimen is already sufficient or adequate.

PD parameters that can be used in the present invention in order todetermine the dosage regime of an active agent of the present inventioninclude, but are not limited to, serum phosphorus, TmP/GFR (renaltubular maximum reabsorption rate of phosphate to glomerular filtrationrate), maximum TmP/GFR, serum 1,25-dihydroxy vitamin D, and serum25-hydroxy vitamin D. In some embodiments, serum phosphorus includes AUCserum phosphorus, e.g., AUC_(last), AUC_(inf), or AUC_(n) within a doseinterval or peak and trough serum phosphorus.

In some embodiments, the dosing regimen of an active agent of thepresent invention is determined based on one or more PD parameters toprovide a sufficient binding agent for any changes in FGF23 levelsduring chronic anti-FGF23 ligand therapy. In some other embodiments, thedosing regimen of an active agent of the present invention is determinedbased on one or more PD parameters to provide an FGF23 level or activitywithin at least 60%, 70%, 80%, 85%, 90%, or 95%, 98%, 100%, 105%, 110%,115%, or 120% of the normal FGF23 level or activity, e.g., of an adultor child. In some other embodiments, the dosing regimen of an activeagent of the present invention is determined based on one or more PDparameters to maintain a steady level or activity of FGF23, e.g., thatdoes not deviate away from the normal FGF23 level or activity for morethan 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 30% of the normalFGF23 level or activity.

In some embodiments, the dosing regimen is determined based on one ormore PD parameters to achieve normalization or stabilization of one ormore PD parameters. In some embodiments, the dosing regimen isdetermined to keep one or more PD parameters as much above the baselinelevel as possible, as long as possible, and/or as stable as possible,without raising any toxicity issue. For example, the dosing regimen isdetermined to keep serum phosphorus at a stable level above baseline,e.g., with minimum or acceptable variability. In yet some otherembodiments, the dosing regimen is determined to keep one or more PDparameters around a predetermined level, e.g., predetermined standardlevel. For example, in one embodiment, the dosing regimen is determinedto keep serum phosphorus at a predetermined standard level, e.g., astable increase of about 0.5 to 1.5 mg/dL above baseline. In anotherembodiment, the dosing regimen is determined to keep serum phosphorus ata predetermined standard level, e.g., a stable increase of about 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3mg/dL above baseline.

In some embodiments, the dosing regimen is determined to provide asustained effect on NaPi transporter, e.g., without a substantialdecline of NaPi transporter activity during a dosing cycle. In someembodiments, the dosing regimen is determined to provide a steady NaPitransporter level or activity, e.g., a NaPi transporter level oractivity that does not deviate away from the normal NaPi transporterlevel or activity for more than, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 15%, 20%, or 30% of the normal NaPi transporter level oractivity. In some embodiments, the dosing regimen is determined toprovide an increase in bone remodeling.

As used herein, when the level of a PD parameter goes towards the levelof a predetermined standard level, it is called normalization. As usedherein, when the level of a PD parameter reduces its variation from thelevel of a predetermined standard level, it is called stabilization.

As used herein, the term “predetermined standard level” refers to levelsobtained from standardized data or data set representing the average,representative features or characteristics of one or more PD parametersproviding effective dosing regimen for a particular anti-FGF23 ligandtherapy in a specific population of subjects. Such features orcharacteristics include, but are not limited to, transcript abundance,transcript stability, transcription rate, translation rate,post-translation modification, protein abundance, protein stability,and/or protein enzymatic activity, etc. In some embodiments, thespecific population of subjects are consisting of about 5, about 10,about 20, about 50, about 100, about 200, about 300, about 400, about500, about 1000, about 5000, about 10K, or more individual subjects. Thepredetermined activity profile can be a standardized data set or a dataset collected before, during, or after the specific population ofsubjects has been all exposed to a drug. In some embodiments, thespecific population is consisting of subjects under effective dosingregimen of an anti-FGF23 ligand.

In some embodiments, the PD parameter is serum phosphorus. In someembodiments, the dosage regimen is determined based on Area Under theCurve (AUC) of serum phosphorus. In some embodiments, the AUC isAUC_(last), AUC_(inf), or AUC_(n). In some embodiments, the PD parameteris the AUC of serum phosphorus within a dosage interval. In someembodiments, the PD parameter is the peak and trough serum phosphorus.In some embodiments, the dosage regimen is determined to maintain aserum phosphorus level during the treatment that is close to, or above apredetermined serum phosphorus level. In some embodiments, the dosageregimen is determined to provide an adequate, stable increase ofphosphorus in the patient during a dosing cycle when compared to thebaseline level in the patient. In some embodiments, the dosage regimenis determined to maintain a relatively stable serum phosphorus levelabove the baseline level in the patient. For example, in someembodiments, a stable increase in serum phosphorus of about 0.5 to about1.5 mg/dL is likely to be sufficient.

In some embodiments, when the patient is an adult, the baseline serumphosphorus level is about 2.5 to about 4.5 mg/dL. In some embodiments,when the patient is a 5-12 year old child, the baseline serum phosphoruslevel is about 2.9 to about 5.7 mg/dL. In some embodiments, when thepatient is an adult, the predetermined serum phosphorus level is about0.5 mg to 1.5 mg/dL higher than the baseline, e.g., of about 2.5 toabout 4.5 mg/dL. In some embodiments, when the patient is a 5-12 yearold child, the predetermined serum phosphorus level is about 0.5 mg to1.5 mg/dL higher than the baseline of about 2.9 to about 5.7 mg/dL. Insome embodiments, depending on the age of the patient, the predeterminedserum phosphorus level is about 2.5 mg/dL, about 2.6 mg/dL, about 2.7mg/dL, about 2.8 mg/dL, about 2.9 mg/dL, about 3.0 mg/dL, about 3.1mg/dL, about 3.2 mg/dL, about 3.3 mg/dL, about 3.4 mg/dL, about 3.5mg/dL, about 3.6 mg/dL, about 3.7 mg/dL, about 3.8 mg/dL, about 3.9mg/dL, about 4.0 mg/dL, about 4.1 mg/dL, about 4.2 mg/dL, about 4.3mg/dL, about 4.4 mg/dL, about 4.5 mg/dL, about 4.6 mg/dL, about 4.7mg/dL, about 4.8 mg/dL, about 4.9 mg/dL, about 5.0 mg/dL, about 5.1mg/dL, about 5.2 mg/dL, about 5.3 mg/dL, about 5.4 mg/dL, about 5.5mg/dL, about 5.6 mg/dL, about 5.7 mg/dL, about 5.8 mg/dL, about 5.9mg/dL, about 6.0 mg/dL, about 6.1 mg/dL, about 6.2 mg/dL, about 6.3mg/dL, about 6.4 mg/dL, about 6.5 mg/dL, about 6.6 mg/dL, about 6.7mg/dL, about 6.8 mg/dL, about 6.9 mg/dL, about 7.0 mg/dL, about 7.1mg/dL, about 7.2 mg/dL, about 7.3 mg/dL, about 7.4 mg/dL, about 7.5mg/dL, about 7.6 mg/dL, about 7.7 mg/dL, about 7.8 mg/dL, about 7.9mg/dL, or more. In some other embodiments, the dosing regimen of anactive agent of the present invention is determined based on one or morePD parameters to provide a serum phosphorus level or activity within atleast 60%, 70%, 80%, 85%, 90%, or 95%, 98%, 100%, 105%, 110%, 115%,120%, or more of the normal serum phosphorus level or activity, e.g., ofan adult or child. In some other embodiments, the dosing regimen of anactive agent of the present invention is determined based on one or morePD parameters to maintain a steady level or activity of serumphosphorus, e.g., that does not deviate away from the normal serumphosphorus level or activity for more than 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 15%, 20%, 30%, or more of the normal serum phosphorus level oractivity.

In some embodiments, the PD parameter is Tmp/GFR (the ratio of the renaltubular maximum reabsorption rate of phosphate to glomerular filtrationrate, see Barth et al., Ann Clin Biochem 2000; 37: 79-81). In someembodiments, the PD parameter is the peak and trough Tmp/GFR. In someembodiments, the dosing regimen is determined to maintain a Tmp/GFRduring the treatment that is close to, or above a predetermined Tmp/GFR.In some embodiments, the dosing regimen is determined to provide anadequate increase of Tmp/GFR in the patient during a dosing cycle whencompared to the baseline level in the patient. In some embodiments, thedosing regimen is determined to maintain a relatively stable Tmp/GFRabove the baseline level in the patient. In some embodiments, thepredetermined Tmp/GFR is about 2.0, about 2.1, about 2.2, about 2.3,about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6,about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9,or more.

In some embodiments, the PD parameter is serum 1,25-dihydroxy vitamin D.In some embodiments, the PD parameter is the peak and trough serum1,25-dihydroxy vitamin D. In some embodiments, the dosing regimen isdetermined with the goal to maintain a serum 1,25-dihydroxy vitamin Dduring the treatment that is close to, similar to, or above apredetermined serum 1,25-dihydroxy vitamin D. In some embodiments, thedosing regimen is determined to provide an adequate increase of serum1,25-dihydroxy vitamin D in the patient during a dosing cycle whencompared to the baseline level in the patient. In some embodiments, thedosing regimen is determined to maintain a relatively stable serum1,25-dihydroxy vitamin D above the baseline level in the patient. Insome embodiments, the predetermined serum 1,25-dihydroxy vitamin D isabout 40 pg/ML, about 45 pg/ML, about 50 pg/ML, about 55 pg/ML, about 60pg/ML, about 65 pg/ML, about 70 pg/ML, about 75 pg/ML, about 80 pg/ML,about 85 pg/ML, about 90 pg/ML, about 95 pg/ML, about 100 pg/ML, about105 pg/ML, about 110 pg/ML, about 115 pg/ML, about 120 pg/ML, about 125pg/ML, about 130 pg/ML, about 135 pg/ML, about 140 pg/ML, about 145pg/ML, about 150 pg/ML, about 155 pg/ML, about 160 pg/ML, about 165pg/ML, about 170 pg/ML, about 175 pg/ML, about 180 pg/ML, about 185pg/ML, about 190 pg/ML, about 195 pg/ML, about 200 pg/ML, or more.

In some embodiments, the PD parameter is serum 25-hydroxy vitamin D. Insome embodiments, the PD parameter is the peak and trough serum25-hydroxy vitamin D. In some embodiments, the dosing regimen isdetermined with the goal to maintain a serum 25-hydroxy vitamin D duringthe treatment that is close to, similar to, or above a predeterminedserum 25-hydroxy vitamin D. In some embodiments, the dosage regime isdetermined to provide an adequate increase of serum 25-hydroxy vitamin Din the patient during a dosing cycle when compared to the baseline levelin the patient. In some embodiments, the dosage regime is determined tomaintain a relatively stable serum 25-hydroxy vitamin D above thebaseline level in the patient. In some embodiments, the predeterminedserum 25-hydroxy vitamin D is about 25 nmol/L, about 26 nmol/L, about 27nmol/L, about 28 nmol/L, about 29 nmol/L, about 30 nmol/L, about 31nmol/L, about 32 nmol/L, about 33 nmol/L, about 34 nmol/L, about 35nmol/L, about 36 nmol/L, about 37 nmol/L, about 38 nmol/L, about 39nmol/L, about 40 nmol/L, about 41 nmol/L, about 42 nmol/L, about 43nmol/L, about 44 nmol/L, about 45 nmol/L, about 46 nmol/L, about 47nmol/L, about 48 nmol/L, about 49 nmol/L, about 50 nmol/L, about 51nmol/L, about 42 nmol/L, about 53 nmol/L, about 54 nmol/L, about 55nmol/L, about 56 nmol/L, about 57 nmol/L, about 58 nmol/L, about 59nmol/L, about 60 nmol/L, about 61 nmol/L, about 62 nmol/L, about 63nmol/L, about 64 nmol/L, about 65 nmol/L, about 66 nmol/L, about 67nmol/L, about 68 nmol/L, about 69 nmol/L, about 70 nmol/L, about 71nmol/L, about 72 nmol/L, about 73 nmol/L, about 74 nmol/L, about 45nmol/L, about 76 nmol/L, about 77 nmol/L, about 78 nmol/L, about 79nmol/L, about 80 nmol/L, or more.

In some embodiments, the dosing regimen comprises administeringanti-FGF23 ligand more frequently than a dosing regimen determined basedon one or more PK parameters. In some other embodiments, the dosingregimen comprises administering anti-FGF23 ligand, e.g., anti-FGF23antibody more frequently than a monthly dosing regimen. For example, thedosing regimen comprises administrating an anti-FGF23 ligand, e.g.,anti-FGF23 antibody about every 3 days, about every 5 days, about everyweek, about every 10 days, about every 2 weeks, about every 17 days,about every three weeks, about every 25 days or more.

According to another aspect of the present invention, it providesmethods for treating FGF23 related disorders via administering one ormore anti-FGF23 ligands according to a dosing regimen determined basedon one or more PD parameters. In some embodiments, the FGF23 relateddisorders include any condition or disorder directly or indirectlyrelated to FGF23 or one or more components of FGF23 signaling pathway.For example, in one embodiment the FGF23 related disorder is XLH or TIO.In some other embodiments, the dosing regimen includes using ananti-FGF23 antibody twice per month, e.g., at a fixed dose.

The present invention also provides methods of treating FGF23 relateddisorders, e.g., via increasing bone remodeling. In some embodiments,the disorders are bone disorders. In some embodiments, the methodscomprise inducing bone remodeling in a patient in need of the treatment.In some embodiments, the bone remodeling is induced by an anti-FGF23ligand, such as an anti-FGF23 antibody. In some embodiments, theadministration provides statistically significant therapeutic effect fortreating the disorder. In some embodiments, the anti-FGF23 ligand causesan increase of a marker in the patient compared to the baseline level ofthe marker. In some embodiments, a marker is selected from the groupconsisting of Serum Type 1 Pro-collagen/N-terminal (P1NP),carboxy-terminal collagen crosslink (CTX), Osteocalcin, BALP, serum CTxand urine NTX/creatine ratio.

In general, bone remodeling involves a series of highly regulated stepsthat depend on the interactions of the mesenchymal osteoblastic lineageand the hematopoietic osteoclastic lineage. The initial “activation”stage involves the interaction of osteoclast and osteoblast precursorcells which leads to the differentiation, migration, and fusion of thelarge multinucleated osteoclasts. Bone remodeling is systematicallyregulated by hormones, including PTH, 1,25-dihydroxy vitamin D, andcalcitonin, and several other systemic hormones such as growth hormone,glucocorticoids, and estrogen. Local regulators of bone remodelinginclude, but are not limited to, cytokines (IL-1, TNF, LI-6, IL-11, ODF,IL-4, IL-13, IL-18, IFN, OPG, and IL-1ra), prostaglandins, osteoclastdifferentiation factor, colony-stimulating factors (e.g., M-CSF andGM-CSF), leukotrienes, nitric oxide, and growth factors (e.g., IGF,TGFβ, FGF, PDGF, and PTHrP).

The present invention also provides methods of treating and/orpreventing a FGF23 related disorder by administering an effective amountof an anti-FGF23 ligand to a subject in need of such a treatment with adosing regimen more frequent than a monthly dosing regimen, such as anevery two weeks dosing regimen. In some embodiments, anti-FGF23 ligandis administered to a subject about every 1, 1.5, 2, 2.5, 3, or 3.5weeks. In some embodiments, the subject has a disorder associated withabnormal FGF23 signaling, such as disorders selected from the groupconsisting of, autosomal dominant hypophosphatemic rickets/osteomalachia(ADHR), X-linked hypophosphatemia (XLH), autosomal recessivehypophosphatemic rickets (ARHR), fibrous dysplasia (FD), McCune-Albrightsyndrome complicated by fibrous dysplasia (MAS/FD), Jansen's metaphysealchondrodysplasia (Jansen's Syndrome), autosomal dominant polycystickidney disease (ADPKD), tumor-induced osteomalacia (TIO), chronicmetabolic acidosis, and ectopic calcification. In some embodiments, thedisorder is ectopic calcification.

The present invention also provides methods of controlling serumphosphate levels. In some embodiments, the methods compriseadministrating an anti-FGF23 ligand to a subject in need of such atreatment. In some embodiments, the methods comprise administrating ananti-FGF23 ligand to a subject with a dosing regimen more frequent thana monthly dosing regimen, such as an every two weeks dosing regimen. Insome embodiments, anti-FGF23 ligand is administered to a subject aboutevery 1, 1.5, 2, 2.5, 3, or 3.5 weeks. In some embodiments, the methodsstabilize or normalize the serum phosphate level in the subject. In someembodiments, the methods minimize the swing of the serum phosphatelevel, for example, to keep the serum phosphate level close to apredetermined level.

The following examples illustrate various aspects of the invention. Theexamples should, of course, be understood to be merely illustrative ofonly certain embodiments of the invention and not to constitutelimitations upon the scope of the invention.

EXAMPLES Example 1—First Phase I/II, Open-Label, Repeat-Dose,Dose-Escalation Study of KRN23 in Adult Subjects with X-LinkedHypophosphatemia

Synopsis:

Investigators and Study Centers: A total of 32 subjects were enrolled at6 sites in the United States and Canada

Clinical Phase of Development: Phase 1/2.

Primary Objective: The primary objective of this study was to assess thesafety and efficacy of repeat-doses of KRN23 subcutaneous (SC)administration in adult subjects with X-linked hypophosphatemia (XLH).

Secondary Objective: The secondary objectives of this study were toevaluate the effects of repeat-doses of KRN23 SC administration onpharmacodynamics (PD), pharmacokinetics (PK), immunogenicity, andquality of life (QoL).

Bone Substudy Objective: To evaluate the effects of repeat-doses ofKRN23 SC administration compared to placebo on bone mineral density andbone quality.

Study Design and Methodology: This was a Phase I/II, open-label,dose-escalation and multicenter study of KRN23 in adult subjects withXLH. The study design consisted of 3 periods: Screening, On-treatment,and Follow-up. During the Screening period, all subjects underwent aScreening visit (the maximum duration of the Screening period was 30days). Following the Screening visit (Visit 1), all eligible subjectswere registered at Baseline (Day 0) into the On-treatment period and thestarting dose of KRN23 0.05 mg/kg SC was administered. Subjects weretreated with up to 4 doses (1 dose every 28 days) of KRN23 usingstepwise dose-escalation from 0.05 mg/kg→0.1 mg/kg→0.3 mg/kg→0.6 mg/kg.Intra-subject dose-escalation was based on serum phosphorus levelsguided by a dose-escalation algorithm and other safety observations(i.e., adverse event [AE] monitoring, safety laboratory parameters,immunogenicity, and physical examination findings). Upon completion ofthe treatment period, subjects underwent a final assessment at theclinic during the Follow-up period (maximum of 10 days).

A bone substudy was planned in a subset of subjects to evaluate theeffects of single-blind KRN23 versus placebo on various bone parameters.Subjects underwent evaluation of additional eligibility criteria forparticipation in this substudy and additional clinical assessments,inclusive of peripheral quantitative computed tomography (pQCT) anddual-energy X-ray absorptiometry (DXA) scan at Baseline and at the endof the study (Day 120). A biopsy of the iliac crest was to be performedfor subjects in this substudy who entered the extension study (secondPhase I/II clinical trial). The Sponsor terminated the bone substudy dueto slow accrual.

Diagnosis: Male and female subjects at least 18 years of age with adocumented clinical diagnosis of XLH and intact fibroblast growth factor23 (FGF23) level >30 pg/mL, renal tubular maximum reabsorption rate ofphosphate to glomerular filtration rate (TmP/GFR)<2.0 mg/dL, eGFR≥60mL/min (using Cockcroft-Gault formula), and corrected serum calciumlevel <10.8 mg/dL (if serum albumin was <4.0 g/dL).

Number of Subjects (planned and analyzed): Approximately 42 subjectswere planned; 33 subjects were screened for enrollment (30 in theopen-label portion of the study; 3 in the bone substudy); 1 of the 3subjects screened in the bone substudy did not meet eligibility criteriaand was discontinued from the study prior to treatment assignment.

TABLE 1 Subject Disposition Open-label Bone Substudy Total KRN23 KRN23Placebo KRN23 N = 30 N = 1 N = 1 N = 31 Enrolled 30 1 1 31 SafetyAnalysis Set^(a) 27 (90.0) 1 (100.0) 1 (100.0) 28 (90.3) EfficacyAnalysis Set^(b) 26 (86.7) 1 (100.0) 1 (100.0) 27 (87.1) Completion ofTherapy 25 (83.3) 1 (100.0) 1 (100.0) 26 (83.9) Primary Reason forWithdrawal Subject Withdrawal of  2 (6.7) 0 0  2 (6.5) Consent^(c)Adverse Event 2  2 (6.7)^(d) 0 0  2 (6.5) (6.7)d 0 0 2 (6.5) Subject DidNot Meet  1 (3.3) 0 0  1 (3.2) Inclusion/Exclusion Criteria^(e) ^(a)Allsubjects who received at least one dose of KRN23 or placebo ^(b)Allsubjects who received at least one dose of KRN23 or placebo and hadcompleted at least one 28-day postdose evaluation. ^(c)Subject 0207 and0212 withdrew consent prior to the first dose of KRN23. ^(d)Subject 0105discontinued due to an adverse event that began prior to dosing andSubject 0206 discontinued due to a TEAE (injection site urticaria).^(e)This subject is a screen failure.

Test Product, Dose and Mode of Administration, Batch Number:

KRN23 for SC injection: KRN23 0.05, 0.1, 0.3 or 0.6 mg/kg administeredSC to the abdomen.

Open-label portion: 10 mg/mL in a 5-mL single-use vial (Batch Number:YU008B).

Bone substudy: 10 mg/mL in a 5-mL single-use vial (Batch Number:YU008E).

Comparative Agent, Dose, and Mode of Administration, Batch Number:

Open-label portion: None

Bone substudy: KRN23 placebo for SC administration to the abdomen (Batchnumber: PYU120413A).

Duration of Treatment: The duration of treatment was up to 110 days. Thetotal duration of the study was approximately 150 days for subjects whoentered the extension study, and was planned to be 195 days for subjectswho did not enter the extension study.

Endpoints:

Efficacy: The primary efficacy endpoint was the number and percentage ofsubjects with postdose serum phosphorus levels ≤2.5 mg/dL; >2.5 mg/dLbut ≤3.5 mg/dL, >3.5 mg/dL but ≤4.5 mg/dL; and >4.5 mg/dL.

Pharmacodynamic: Secondary PD endpoints included the changes fromBaseline in:

-   -   Serum phosphorus; the area under the concentration time curve        (AUC) from Baseline; the time interval of serum phosphorus        levels >2.5 mg/dL.    -   TmP/GFR and serum 1,25-dihydroxy vitamin D [1,25(OH)₂D].    -   Serum 25-hydroxy vitamin D [25(OH) D], total calcium, ionized        calcium, calcitonin, intact parathyroid hormone (iPTH); 2-hour        urinary measures of tubular reabsorption of phosphate (TRP),        calcium/creatinine ratio, fractional excretion of calcium        (FECa); and 24-hour urinary measures of phosphorus, calcium,        creatinine, and calcium/creatinine ratio.    -   Sex hormones: estradiol, testosterone, free testosterone and sex        hormone binding globulin (SHBG).    -   Bone formation biomarkers (bone alkaline phosphatase [BALP],        procollagen type 1 N-propeptide [P1NP], and osteocalcin) and        bone resorption biomarkers (carboxy-terminal cross-linked        telopeptide of type 1 collagen [CTx]) and osteocalcin and amino        terminal cross-linked telopeptide of type 1 collagen (urinary        NTx).        Pharmacokinetic:    -   Characterize the PK parameters for KRN23 following multiple-dose        SC administration.    -   Characterize the population PK of KRN23 dose levels from        cumulative dosing.        Quality of Life:    -   Changes from Baseline in patient-reported outcomes (PROs) based        on the Study 36-item Short Form, Version 2 (SF-36v2) and Western        Ontario and McMaster Osteoarthritis Index (WOMAC).        Safety: Safety included the number and percentage of subjects        with adverse events, by severity and relationship to study drug,        and the clinically significant changes from Baseline in clinical        laboratory evaluations (hematology, chemistry, and urinalysis),        vital signs, physical and neurological examinations, cardiac        computed tomography (CT)/coronary calcium scoring and aortic        calcium scoring, renal ultrasound, electrocardiogram (ECG); and        anti-KRN23 antibody assessment. In addition, changes in bone        parameters in the forearm and tibia, as measured by pQCT, and        changes in bone mineral density and other bone parameters in the        lumbar spine and proximal femur, as measured by DXA scan, were        assessed in the bone substudy.

Statistical Methods: Absolute and change from Baseline for continuousdata were summarized descriptively including the number of observations,mean, standard deviation, median, minimum and maximum. Categoricalvariable were summarized using frequency counts and percentages. Alldata summaries were presented for the following treatment groups:open-label treatment (KRN23), single-blind treatment in the bonesubstudy (KRN23 and placebo) and total KRN23. Pharmacodynamic parametersmeasured included serum phosphorus levels, TmP/GFR, and serum 1,25(OH)₂Dalong with other biochemical parameters. Area under the curve for thechange from baseline in these pharmacodynamic parameters were calculatedfor the 120 days of treatment or up to last measured time point beforewithdrawal (AUC_(last)) and for each of 4 dosing intervals (AUC1, AUC2,AUC3 and AUC4), where AUCn was the AUC from baseline (i.e., before firstdose) for the dosing interval “n”. The number and percentage of subjectsreporting treatment-emergent adverse events (TEAEs) were evaluatedacross treatment arms.

Pharmacokinetic and PK-PD Methods: Descriptive statistics were used forserum KRN23 concentration data and PK parameters. Serum KRN23 sampleswere analyzed using non-compartmental methods. PK parameters (area underthe serum concentration time curve from zero to last measuredconcentration [AUClast], area under the serum concentration-time curvefrom zero to infinity [AUCinf], apparent volume of distribution [V/F],apparent clearance [CL/F], and terminal elimination half-life after 4thdosing interval [t½]) were estimated using subject serum concentrationsfor all accumulated doses from Days 1 to 120. In addition, during eachdosing interval n, area under the serum concentration-time curve forn-th dosing interval [AUCn], maximum serum concentration [Cmax, n],predose serum concentration (Cmin, n), and time to peak plasmaconcentration [tmax, n] were estimated. Scatter plots were generated toexplore the PK-PD correlation using AUCn and AUClast for PK and PDmeasures. Concentration (serum KRN23) and effect (serum phosphorusincrease from baseline) relationship was described by an empirical Emaxmodel.

Overall study design and plan: This Phase I/II, open-label,dose-escalation multicenter study planned to enroll approximately 42male and female adult subjects with XLH. Of the 42 subjects, 18 wereplanned to be in bone substudy. Subjects participating in the open-labelportion of the study received up to four doses of KRN23 (0.05, 0.1, 0.3,and 0.6 mg/kg) administered subcutaneously (SC) every 28-days over a120-day period. All subjects received the starting dose of KRN23 0.05mg/kg SC. Intra-subject step-wise dose escalation of KRN23 was based onserum phosphorus levels guided by a dose-escalation algorithm and safetyevaluations (i.e. AE monitoring, safety laboratory parameters,immunogenicity, and physical examination findings). Subjectsparticipating in the bone substudy were randomized to eithersingle-blind KRN23 or Placebo and received up to four doses of KRN23(0.05, 0.1, 0.3, and 0.6 mg/kg) administered SC every 28-days over a120-day period.

Study Medications: Subjects received KRN23 via SC injection(s) to theabdomen (avoiding a 1-inch radius surrounding the umbilicus) on dosingdays: Days 0, 28, 56 and 84, using stepwise dose-escalation from 0.05mg/kg→0.1 mg/kg→0.3 mg/kg→0.6 mg/kg. Subjects returned on Days 3, 7, 12,18 and 26 of each dosing cycle (4 dosing cycles) for scheduled clinical,laboratory, PK and PD assessments. All the visits were conducted in afasted state (i.e., refrain from eating and drinking, other than water)for at least 8-hours prior to the study visit.

Disease Characteristics at baseline: For subjects in the efficacyanalysis, Baseline disease characteristics including the levels of serumphosphorus, TmP/GFR, 1,25(OH)2D, and intact FGF23 for all subjects inthe Efficacy Analysis Set are presented in the table below. Overall,mean serum phosphorus and TmP/GFR levels at Baseline were below thelower limit of the normal reference range. At Baseline, the overall mean1,25(OH)₂D level was within the normal reference range for all subjects.Baseline FGF23 levels ranged from 52.8 to 4620 pg/mL.

TABLE 2 Baseline Disease Characteristics (Efficacy Analysis Set)Open-label Treatment Bone Substudy Total KRN23 KRN23 Placebo KRN23Characteristic N = 26 N = 1 N = 1 N = 27 FGF23 (pg/mL) n 26 1 1 27 Mean(SD) 291.2 (886.24) 52.8 (NA) 86.6 (NA) 282.4 (870.24) Min, Max (56.4,4620.0) (52.8, 52.8) (86.6, 86.6) (52.8, 4620.0) Serum phosphorus(mg/dL) n 26 1 1 27 Mean (SD) 1.89 (0.34) 2.0 (NA) 1.6 (NA) 1.89 (0.33)Min, Max (1.2, 2.8) (2.0, 2.0) (1.6, 1.6) (1.2, 2.8) TmP/GFR (mg/dL)^(a)n 26 1 1 27 Mean (SD) 1.61 (0.37) 1.45 (NA) 1.25 (NA) 1.60 (0.36) Min,Max (0.84, 2.26) (1.45, 1.45) (1.25, 1.25) (0.84, 2.26) Serum 1,25(OH)₂D(pg/mL) n 23 1 1 24 Mean (SD) 36.3 (14.52) 42.0 (NA) 67.0 (NA) 36.6(14.25) Min, Max (10, 62) (42, 42) (67, 67) (10, 62) Serum total calcium(mg/dL) n 26 1 1 27 Mean (SD) 9.10 (0.38) 9.30 (NA) 9.30 (NA) 9.11(0.38) Min, Max (8.5, 10.2) (9.3, 9.3) (9.30, 9.30) (8.5, 10.2) SerumiPTH (pg/mL) n 26 1 1 27 Mean (SD) 82.2 (32.74) 74.0 (NA) 77.0 (NA) 81.9(32.15) Min, Max (38, 143) (74, 74) (77, 77) (38, 143) BALP (μg/L) n 261 1 27 Mean (SD) 28.1 (12.96) 34.4 (NA) 20.7 (NA) 28.3 (12.77) Min, Max(8.2, 52.4) (34.4, 34.4) (20.7, 20.7) (8.2, 52.4) 24-hr urine calcium(mg/24 hr) n 26 1 1 27 Mean (SD) 91.4 (65.66) 30.0 (NA) 54.0 (NA) 89.1(65.46) Min, Max (11, 253) (30, 30) (54, 54) (11, 253) 24-hr urinecreatinine (g/24 hr) n 26 1 1 27 Mean (SD) 1.31 (0.62) 1.23 (NA) 1.04(NA) 1.31 (0.61) Min, Max (0.54, 3.01) (1.23, 1.23) (1.04, 1.04) (0.54,3.01) 2-hr calcium/creatinine ratio, (mg/g creatinine) n 26 1 1 27 Mean(SD) 49.4 (38.59) 7.0 (NA) 36.0 (NA) 47.8 (38.71) Min, Max (11, 192) (7,7) 36, 36) (7, 192) 1,25(OH)₂D = 1,25-dihydroxyvitamin D; BALP = bonealkaline phosphatase; FGF23 = fibroblast growth factor 23; hr = hour;iPTH = intact parathyroid hormone; Max = maximum; Min = minimum; NA =not applicable; SD = standard deviation; TmP/GFR = renal tubular maximumreabsorption rate of phosphate to glomerular filtration rate. ^(a)Normalrange: 2.5-4.3 for TmP/GFR?

Pharmacokinetic/Pharmacodynamic Sampling: Blood samples for theassessment of PK of KRN23 were collected from all subjects according tothe sampling schedule shown in Table 3 below. Serum samples wereanalyzed for KRN23 concentrations using validated bioanalytical methods.

TABLE 3 Pharmacokinetic Sampling Schedule Dosing Cycle Day relative todose* PK Sample Time 1 0 Pre-dose 3 At clinic visit 7 At clinic visit 12At clinic visit 18 At clinic visit 26 At clinic visit 2 28 or Day 0 for2nd dosing cycle Pre-dose 3 At clinic visit 7 At clinic visit 12 Atclinic visit 18 At clinic visit 26 At clinic visit 3 56 or Day 0 for 3rddosing cycle Pre-dose 3 At clinic visit 7 At clinic visit 12 At clinicvisit 18 At clinic visit 26 At clinic visit 4 84 or Day 0 for 4th dosingcycle Pre-dose 3 At clinic visit 7 At clinic visit 12 At clinic visit 18At clinic visit 26 At clinic visit Follow up visit/ 120 or Day 36 for4th dosing At clinic visit Early withdrawal cycle/Early withdrawal *±3days window for each visit

Samples for the assessment of PD parameters were collected on dosingdays (Days 0, 28, 56 and 84), on Days 3, 7, 12, 18 and 26 of each dosingcycle, and Day 120 (follow-up visit/early withdrawal). In addition,blood samples for measurements of bone biomarkers were collected atbaseline (Day 0), at predose time in each dosing cycle (Days 28, 56 and84) and at the follow-up visit (Day 120).

Drug Concentration Measurements: Serum samples were analyzed for KRN23at Kyowa Hakko Kirin California, Inc. (KKC) formerly Kirin Pharma USA,Inc., using a validated sandwich Enzyme Linked Immunosorbant Assay(ELISA) method. The lower limit of quantification (LLOQ) for the KRN23assay was 50 ng/mL. The standard curve had a range of 50-3000 ng/mL forKRN23. All blood and urine samples for PD parameters were analyzed at acentral laboratory using available commercial assay kits and/orvalidated assay methods.

SUMMARY OF RESULTS

Exposure: A total of 28 subjects were treated with KRN23 (27 in theopen-label portion of the study and 1 in the bone substudy) and 1subject was treated with placebo in the bone substudy. The mean totaldose of KRN23 administered was 0.05±0.00 mg/kg, 0.10±0.01 mg/kg,0.28±0.06 mg/kg, and 0.48±0.16 mg/kg during dosing intervals 1, 2, 3,and 4, respectively. One subject in the placebo group in the bonesubstudy received all 4 doses of placebo.

Study Population: Among the 28 subjects treated with KRN23, the medianage was 41.9 years (range: 19 to 66 years); the majority (67.9%; 19/28)were women, and nearly all (96.4%; 27/28) were white/Caucasian and 1 wasBlack/African American/African Caribbean. The median height of subjectstreated with KRN23 was 149.7 cm (range: 121.9 to 170.2 cm) and medianweight was 70.1 kg (range: 46.4 to 121.9 kg). The median body mass indexof subjects treated with KRN23 was 30.7 kg/m2 (range 19.7 to 67.6kg/m2). The overall, mean serum phosphorus and TmP/GFR levels atBaseline were below the lower limit of the normal reference range. AtBaseline, the overall mean 1,25(OH)₂D level was within the normalreference range for subjects treated with KRN23. Baseline FGF23 levelsranged from 52.8 to 4620.0 pg/mL for subjects treated with KRN23.

Efficacy Results: The proportion of KRN23-treated subjects with serumphosphorus levels >2.5 to ≤3.5 mg/dL increased from 3.7% at Baseline(Day 0 in the first dosing interval) to 14.8%, 37.0%, 74.1%, and 70.4%on Day 7 (the time of maximum effect) in the first, second, third, andfourth dosing intervals, respectively. Serum phosphorus levels did notexceed 4.5 mg/dL in any subject at any time point.

Pharmacodynamic Analyses:

Actual sampling times were used for the calculation of the PKparameters. PK parameters for each dosing cycle were estimated based onobserved PK profiles in that cycle and the PK parameters for all doseswere estimated based on total dose received during the 4 dosing cycles.The following PK parameters were determined for KRN23 from the serumconcentration-time data by non-compartmental analysis method:

Parameter Definition AUC_(last) Area under the serum concentration-timecurve from time 0 to the time of last measurable concentration over allthe dosing cycles (Day 120 or early withdrawal) determined using thelinear trapezoidal rule AUC_(inf) Area under the serumconcentration-time curve from time 0 to infinity over all the dosingcycles determined using the linear trapezoidal rule AUC_(n) Area underthe serum concentration-time curve during n-th dosing cycleAUC_(%Extrap) Percentage of AUC inf based on extrapolation C_(max,n)Maximum observed serum concentration during n-th dosing cycle C_(min,n)Predose observed serum concentration at the end of n-th dosing cyclet_(max,n) Time of maximum observed serum concentration during n-thdosing cycle t_(1/2) Terminal elimination half-life after 4th dosingcycle calculated as 1n2/λz where λz is the rate constant associated withelimination phase CL/F Apparent serum clearance calculated as total Doseduring 120 days of treatment/AUC_(inf) V_(z)//F Apparent volume ofdistribution based on terminal elimination phase calculated as CL/F/λzafter 4th dosing cycleFor purpose of AUC computations, missing serum levels were not imputedby interpolation between adjacent points. All serum concentrations belowthe lower limit of quantification of the assay were treated as zero.The AUC₁, AUC₂, AUC₃ and AUC₄ were estimated in Dosing Cycle 1 (Day 0 toDay 28), Dosing Cycle 2 (Day 28 to Day 56), Dosing Cycle 3 (Day 56 toDay 84), and Dosing Cycle 4 (Day 84 to Day 120), respectively.PK analyses for serum KRN23 concentrations were conducted usingnoncompartmental analysis method with Phoenix™ WinNonlin® (Version 6.3,Pharsight—A Certara™ Company, Mountain View, Calif.). Graphical displayswere produced by SigmaPlot (Systat Software, Inc., Point Richmond,Calif.).

Pharmacodynamic Results:

-   -   Mean KRN23 dose increased with each successive dose from 0.05        mg/kg SC for the first dose (starting dose per protocol) to        0.48±0.16 mg/kg for the fourth dose.    -   Mean serum phosphorus achieved maximum levels by Day 7 after        each dose then declined and did not reach pre-dose levels prior        to subsequent doses. Increases in serum phosphorus were        clinically meaningful in magnitude, particularly after the third        and fourth doses.    -   Peak and trough (pre-dose) mean serum phosphorus levels as well        as AUCn increased with successive doses. The mean maximum serum        phosphorus levels increased from 2.21±0.33 mg/dL after the first        dose to 3.03±0.42 mg/dL after the fourth dose.    -   TmP/GFR results followed the same pattern as that observed for        serum phosphorus. The mean maximum TmP/GFR after the first dose        was 1.93±0.45 mg/dL; this increased to 2.97±0.67 mg/dL after the        fourth dose. The magnitude of increase in TmP/GFR was similar to        those in serum phosphorus and was clinically meaningful.    -   Mean serum 1,25(OH)₂D levels increased to maximum levels between        Days 3 and 7 after each dose and then returned to near the        baseline level prior to the next dose. Otherwise, the patterns        observed were the same as those seen with serum phosphorus and        TmP/GFR.    -   There was no correlation between baseline levels of FGF23 and        serum phosphorus. Similarly, increases in serum phosphorus were        not correlated with baseline FGF23 levels.    -   No meaningful effect of KRN23 treatment was observed on the mean        changes from Baseline for the other serum and urine PD        parameters. Mean values for other serum and urine PD parameters        were unchanged after KRN23 dosing compared to Baseline. Serum        parameters included 25(OH)D, total calcium, ionized calcium,        calcitonin, iPTH, estradiol, free testosterone, total        testosterone, and SHBG. Urine markers included 2-hour measures        of TRP, calcium/creatinine ratio, and FECa, and 24-hour measures        of phosphorus, calcium, creatinine, and calcium/creatinine        ratio.    -   Serum markers of bone formation (BALP, P1NP, and osteocalcin)        and bone resorption (CTx and NTx/creatinine) tended to increase        after KRN23 dosing.

Pharmacodynamic Analyses: Briefly, the area under the curve (AUC) forthe change from baseline was calculated over the 120 days of treatmentor up to last measured time point before withdrawal (AUC_(last)) andover each of 4 dosing cycles (AUC₁, AUC₂, AUC₃ and AUC₄) for each of thefollowing PD parameters:

-   -   Calcium homeostasis: albumin, ionized calcium, corrected calcium    -   PD parameters: calcitonin, intact PTH, serum creatinine, serum        phosphorus, serum total calcium    -   Vitamin D: 1,25-dihydroxy Vitamin D (1,25(OH₂)D), 25-hydroxy        Vitamin D (25 (OH)D)    -   Bone biomarkers: bone alkaline phosphatase, c-telopeptide (CTX),        n-telopeptide (NTX), NTX/creatinine ratio, osteocalcin,        procollagen 1 (P1NP)    -   Sex hormones: estradiol, testosterone, free testosterone and sex        hormone binding globulin (SHBG)    -   24-hr urine: calcium, calcium/creatinine ratio, creatinine,        phosphate    -   2-hr urine: creatinine, random calcium, calcium/creatinine        ratio, fractional excretion of calcium (FECa), phosphorous,        tubular reabsorption of phosphate (TRP), Tmp/GFR calculated        For PD parameters, AUC₁, AUC₂, AUC₃ and AUC₄ were estimated in        Dosing Cycle 1 (Day 0 to Day 28), Dosing Cycle 2 (Day 28 to Day        56), Dosing Cycle 3 (Day 56 to Day 84), and Dosing Cycle 4 (Day        84 to Day 120), respectively. Serum PD AUC_(last) was estimated        from Day 0 to Day 120 or early withdrawal. If predose samples        were not taken for n-th dosing interval, the last sample from        previous dosing interval was used as predose sample.

Pharmacokinetic and PK-PD Relationships:

-   -   The PK exposures (Cmax, n, Cmin, n, AUC_(n) values) increased in        a dose proportional manner with similar mean tmax values across        the 4 dosing intervals. The terminal t½ estimated after the        fourth dosing interval was 16.4±5.8 days.    -   Serum KRN23 and phosphorus concentrations reached maximum levels        at approximately the same time point (approximately Day 7) after        each dose and their concentrations increased and decreased in        parallel, supporting a direct PK-PD relationship described by an        empirical Emax model. The maximum effect of KRN23 on increase in        serum phosphorus levels (Emax) was 1.788 mg/dL and the KRN23        concentration reaching 50% of Emax (EC50) was 2742 ng/mL.    -   The AUCn or AUClast for the change from Baseline in serum        phosphorus, TmP/GFR and 1,25(OH)2D increased linearly with        increasing KRN23 PK exposure (AUCn or AUClast).    -   No meaningful effect of KRN23 treatment was observed on AUCn or        AUClast for change from Baseline for the other PD parameters        (serum albumin, ionized calcium, corrected calcium, calcitonin,        iPTH, creatinine, total calcium, 25(OH)D, estradiol, free        testosterone, total testosterone, SHBG; and 2-hour urine TRP,        calcium/creatinine ratio, and FECa; and 24-hour urine calcium,        calcium/creatinine ratio, creatinine, and phosphate.    -   The AUC for bone formation markers (BALP, P1NP, and osteocalcin)        and bone resorption markers (CTx and NTx/creatinine ratio)        change from Baseline versus KRN23 AUC showed positive trends of        increase following KRN23 treatment.

Quality of Life: Using 2 PRO instruments (SF-36v2 and WOMAC), KRN23treatment resulted in meaningful improvements in the health status ofsubjects with XLH:

-   -   All 10 SF-36v2 measures showed improvements at the end of        treatment compared with Baseline with statistically significant        improvements observed for 3 measures (Role Limitations due to        Physical Health [RP], Bodily Pain [BP], and Physical Component        Summary [PCS]; p<0.05). When corrected for multiplicity, only        the RP was statistically significant (pm<0.05).    -   All 3 WOMAC measures showed improvements at the end of treatment        compared with Baseline with statistically significant        improvements observed for 2 measures (Physical Functioning and        Stiffness; p<0.05).    -   Compared with the general US population, the study population        exhibited greater deficits in baseline BP, Physical Functioning        (PF), RP, and PCS scores. At the end of the study, the deficits        in RP were eliminated (p<0.05) and deficits in PF, BP and PCS        had lessened. All other scores showed improvements toward better        health (Social Functioning [SF], Role Limitations due to        Emotional Problems [RE], Vitality [VT], Mental Health [MH],        Mental Component Summary [MCS], and General Health [GH]).    -   Compared to patients with osteoarthritis, baseline burden of        disease measures for the study population were similar for 8        scales (RP, RE, BP, PF, PCS, VT, MH, and MCS) and were        significantly better for GH and SF. At the end of the study, all        scores improved with statistically significant (p<0.05 and        pm<0.05) improvements observed for RP for the study population        compared with patients with osteoarthritis.    -   The changes in QoL scores correlated moderately (threshold of        0.3 for the correlation) with the following clinical measures:        -   The change from Baseline in BP, GH and Stiffness scores            correlated moderately KRN23 PK exposure (AUClast);        -   The change from Baseline in BP and RE scores correlated            moderately with serum phosphorus and TmP/GFR change from            Baseline (AUClast);        -   The change from Baseline in Pain scores correlated            moderately with 1,25(OH)2D change from Baseline (AUClast).

Safety Results:

-   -   KRN23 was well tolerated following SC administration of 4        escalating doses in this subject population.    -   There were no deaths or SAEs. One subject was withdrawn from the        study because of a TEAE (injection site urticaria). This event        was noted on Day 57 after the third dose of KRN23, was        considered of moderate intensity, and probably related to study        drug. The subject was treated with prednisone and        diphenhydramine.    -   Nearly all subjects (25 subjects, 89.3% treated with KRN23 and        the 1 subject treated with placebo) had at least 1 TEAE during        the study. The most common (reported for at least 5 subjects)        TEAEs for subjects treated with KRN23 were nasopharyngitis (8        subjects, 28.6%, each), arthralgia (7 subjects, 25.0%), and        diarrhea, back pain, and restless legs syndrome (5 subjects,        17.9%, each).    -   Treatment-related TEAEs were reported for 10 subjects (35.7%)        treated with KRN23; no treatment-related TEAEs were reported for        the 1 subject in the placebo group. Diarrhea was the only        treatment-related TEAE reported for more than 1 KRN23-treated        subject (2 subjects, 7.1%). All TEAEs were considered to be mild        or moderate in intensity, except for 2 KRN23-treated subjects (1        with severe myalgia and 1 with severe post-traumatic pain). No        life-threatening or fatal TEAEs were reported.    -   There was no observable pattern of laboratory abnormalities        during the study. Three subjects did have laboratory        abnormalities considered by the Investigator to be TEAEs        (decreased neutrophil count, iron deficiency anemia, increased        blood creatine phosphokinase); however, these were not        considered clinically significant, did not require treatment,        and did not result in discontinuation of the subjects from the        study.    -   No changes were observed in patterns of other safety parameters        obtained (vital signs, physical and neurological examination        findings, cardiac CT scans, renal ultrasounds, and        electrocardiograms) that were suggestive of a treatment effect.    -   No subject developed anti-KRN23 antibodies after dosing. No        positive anti-KRN23 antibody was detected for 1 subject who        experienced injection site reaction (urticaria). No other        subjects experienced any hypersensitivity reactions.    -   There was no evidence of increased serum calcium concentrations,        urinary excretion of calcium, nephrocalcinosis, or coronary        artery calcium scores following KRN23 treatment.

Conclusions: Overall, the sustained effects of KRN23 on serumphosphorus, TmP/GFR, and serum 1,25(OH)₂D levels during a dosinginterval; improvement in QoL scores after 4 doses; direct PK-PDrelationship for serum KRN23 concentration and increase in serumphosphorus levels; and the favorable safety profile in adult subjectswith XLH in this study suggest a potential utility of KRN23 in patientswith XLH.

Analysis of Efficacy, Pharmacodynamic, and Pharmacokinetic Parameters ofKRN23 in Adult Subjects with X-Linked Hypophosphatemia

Primary Efficacy—Serum Phosphorus Levels

The subjects have maximum serum phosphorus levels of ≤2.5 mg/dL, >2.5 to≤3.5 mg/dL, >3.5 to ≤4.5 mg/dL, or >4.5 mg/dL. At Baseline (Day 0 of thefirst dosing interval), nearly all (26 subjects, 96.3%) subjects treatedwith KRN23 had serum phosphorous levels below 2.5 mg/dL; 1 subject hadlevels >2.5 to ≤3.5 mg/dL:

TABLE 4 Proportion of Subject Treated with KRN23 with Serum PhosphorusLevels by Categories (Efficacy Analysis Population) Number (%) ofSubject Treated with KRN23, N = 27 Categories of Serum Visit Day/Phosphorus Levels (mg/dL) Relative Day <2.5 >2.5 to <3.5 >3.5 to<4.5 >4.5 Dosing Interval 1 Visit 2 (Day 0)/0 26 (96.3)  1 (3.7) 0 0Visit 4 (Day 7)/7 23 (85.2)  4 (14.8) 0 0 Visit 7 (Day 26)/26 26 (96.3) 1 (3.7) 0 0 Dosing Interval 2 Visit 8 (Day 28)/0 26 (96.3)  1 (3.7) 0 0Visit 10 (Day 35)/7 17 (63.0) 10 (37.0) 0 0 Visit 13 (Day 54)/26 25(92.6)  2 (7.4) 0 0 Dosing Interval 3 Visit 14 (Day 56)/0 23 (85.2)  2(7.4) 0 0 Visit 16 (Day 63)/7  7 (25.9) 20 (74.1) 0 0 Visit 19 (Day82)/26 19 (70.4)  8 (29.6) 0 0 Dosing Interval 4 Visit 20 (Day 0)/0 18(66.7)  6 (22.2) 0 0 Visit 22 (Day 91)/7  3 (11.1) 19 (70.4) 4 (14.8) 0Visit 25 (Day 110)/26 14 (51.9) 12 (44.4) 0 0

During the first 120 days of the study, the mean total time with serumphosphorus level >2.5 mg/dL for subjects treated with KRN23 was27.8±17.9 days. For 1 subject treated with placebo in the bone substudy,serum phosphorus levels remained essentially unchanged from Baseline(1.6 mg/dL) to the end of the study (Visit 26) (2.0 mg/dL) with postdoselevels ranging from 1.5 to 2.1 mg/dL.

During each dosing interval, the maximum serum phosphorus level wasachieved on Day 7:

TABLE 5 Mean (±SD) Serum Phosphorus Levels and AUC of Serum PhosphorusChange from Baseline for Subjects Treated with KRN23 (Efficacy AnalysisSet) AUC_(n) or AUC_(last) Serum Phosphorus Levels of Serum PhosphorusDosing Day 0 (Predose) Time to Reach Maximum Level Change from BaselineInterval^(a) Mean (SD), mg/dL Maximum Level^(b) Mean (SD), mg/dL Mean(SD), (mg · day/dL) 1 1.89 (0.33), n = 27 Day 7 2.21 (0.33), n = 27 5.68 (5.04), n = 27 2 1.95 (0.36), n = 27 Day 7 2.47 (0.35), n = 2710.72 (6.53), n = 27 3 2.13 (0.27), n = 25 Day 7 2.86 (0.40), n = 2719.56 (9.61), n = 27 4 2.27 (0.32), n = 24 Day 7 3.03 (0.42), n = 2628.20 (12.43), n = 26 Total NA NA NA 63.59 (31.58), n = 27 AUC_(n) =area under the concentration-time curve for the change from Baseline inserum phosphorus concentration during the nth dosing interval (n = 1, 2,3, or 4); AUC_(last) = AUC for the change for Baseline in serumphosphorus concentration from Day 0 to Day 120 (or early withdrawal); NA= not applicable; SD = standard deviation. ^(a)Dosing Interval 1 = Visit2 to Visit 8; Dosing Interval 2 = Visit 8 to Visit 14; Dosing Interval 3= Visit 14 to Visit 20; Dosing Interval 4 = Visit 20 to Visit 26; Total= Visit 2 to Visit 26. ^(b)Time to reach maximum mean serum phosphoruslevel relative to Day 0 of each dosing interval.The proportions of KRN23-treated subjects with serum phosphoruslevels >2.5 to ≤3.5 mg/dL on Day 7 increased from 14.8% in DosingInterval 1 to 37.0%, 74.1%, and 70.4% in Dosing Intervals 2, 3, and 4,respectively.

On Day 91 (Day 7 of the fourth dosing internal), the majority (19subjects, 70.4%) of subjects treated with KRN23 had serum phosphoruslevels >2.5 to ≤3.5 mg/dL; 3 subjects (11.1%) had levels below 2.5 mg/dLand 4 (14.8%) had levels >3.5 to ≤4.5 mg/dL. By Day 110

(Day 26 of the fourth dosing interval), 12 (44.4%) subjects had serumphosphorus levels >2.5 to ≤3.5 mg/dL and 14 (51.9%) had levels below 2.5mg/dL. Serum phosphorus levels did not exceed 4.5 mg/dL at any timepoint in any subject.

In summary, the mean maximum effect of KRN23 occurred on Day 7 afterfourth dosing interval. A total of 23 subjects (85.2%) had serumphosphorus levels >2.5 mg/dL and ≤4.5 mg/dL.

Pharmacodynamic Results

Serum Phosphorus

Mean serum phosphorus levels are summarized in table 6 below anddisplayed graphically in FIG. 1.

TABLE 6 Mean (±SD) Serum Phosphorus Levels and AUC of Serum PhosphorusChange from Baseline for Subjects Treated with KRN23 (Efficacy AnalysisSet) AUC_(n) or AUC_(last) Serum Phosphorus Levels of Serum PhosphorusDosing Day 0 (Predose) Time to Reach Maximum Level Change from BaselineInterval^(a) Mean (SD), mg/dL Maximum Level^(b) Mean (SD), mg/dL Mean(SD), (mg · day/dL) 1 1.89 (0.33), n = 27 Day 7 2.21 (0.33), n = 27 5.68 (5.04), n = 27 2 1.95 (0.36), n = 27 Day 7 2.47 (0.35), n = 2710.72 (6.53), n = 27 3 2.13 (0.27), n = 25 Day 7 2.86 (0.40), n = 2719.56 (9.61), n = 27 4 2.27 (0.32), n = 24 Day 7 3.03 (0.42), n = 2628.20 (12.43), n = 26 Total NA NA NA 63.59 (31.58), 11 = 27 AUC_(n) =area under the concentration-time curve for the change from Baseline inserum phosphorus concentration during the nth dosing interval (n = 1, 2,3, or 4); AUC_(last) = AUC for the change for Baseline in serumphosphorus concentration from Day 0 to Day 120 (or early withdrawal); NA= not applicable; SD = standard deviation. ^(a)Dosing Interval 1 = Visit2 to Visit 8; Dosing Interval 2 = Visit 8 to Visit 14; Dosing Interval 3= Visit 14 to Visit 20; Dosing Interval 4 = Visit 20 to Visit 26; Total= Visit 2 to Visit 26. ^(b)Time to reach maximum mean serum phosphoruslevel relative to Day 0 of each dosing interval.

Mean KRN23 doses were 0.05±0.0, 0.10±0.01, 0.28±0.06, and 0.48±0.16mg/kg for dosing intervals 1, 2, 3, and 4, respectively. During each ofthe 4 dosing intervals, mean serum phosphorus concentrations increasedto maximum concentrations by Day 7 and then declined prior to the nextdosing; both pre-dose and postdose levels increased up as the number ofdoses increased during the 120-day treatment period. As the dosesincreased from the first to the fourth dosing interval, the maximum meanserum phosphorus concentration increased from 2.21±0.33 mg/dL to3.03±0.42 mg/dL, the mean serum phosphorus concentration prior to eachdosing increased from 1.89±0.33 to 2.27±0.32 mg/dL, and mean AUCnincreased from 5.68±5.04 to 28.20±12.43 mg·day/dL.

Peak and trough fluctuations of serum phosphorus were low during eachdosing interval. In the fourth dosing interval, mean serum phosphorusconcentrations fluctuated between a peak (3.03±0.42 mg/dL) and trough(2.27±0.32 mg/dL) with a 0.76 mg/dL difference (33%). The inter-subjectvariability was also low for serum phosphorus concentration. At thefourth dosing interval, between-subject variation of serum phosphoruslevels was only 14% for both trough and peak concentrations(=SD/mean×100%; 0.42/3.03×100%).

Renal Tubular Maximum Reabsorption Rate of Phosphate to GlomerularFiltration Rate

Mean TmP/GFR values for subjects treated with multiple doses of KRN23are summarized in the table 7 below and displayed graphically in FIG. 2.

TABLE 7 Mean (±SD) TmP/GFR Levels and AUC of TmP/GFR Change fromBaseline in Subjects Treated with KRN23 (Efficacy Analysis Set) AUC_(n)or AUC_(last) TmP/GFR Levels of TmP/GFR Change Dosing Day 0(Predose)^(b) Time to Reach Maximum Level from Baseline Interval^(a)Mean (SD), mg/dL Maximum Level^(c) Mean (SD), mg/dL Mean (SD), (mg ·day/dL) 1 1.60 (0.36), n = 27 Day 7 1.93 (0.45), n = 25  5.23 (6.12), n= 27 2 1.68 (0.35), n = 27 Day 7 2.29 (0.47), n = 27  8.79 (6.67), n =27 3 1.79 (0.35), n = 27 Day 7 2.63 (0.61), n = 27 17.66 (9.48), n = 274 2.05 (0.34), n = 27 Day 7 2.97 (0.67), n = 26 28.55 (13.40), n = 26Total NA NA NA 68.42 (37.96), n = 27 AUC_(n) = area under theconcentration-time curve for the change from Baseline in TmP/GFR duringthe nth dosing interval (n = 1, 2, 3, or 4); AUC_(last) = AUC for thechange for Baseline in TmP/GFR from Day 0 to Day 120 (or earlywithdrawal); NA = not applicable; SD = standard deviation; TmP/GFR =renal tubular maximum reabsorption rate of phosphate to glomerularfiltration rate. ^(a)Dosing Interval 1 = Visit 2 to Visit 8; DosingInterval 2 = Visit 8 to Visit 14; Dosing Interval 3 = Visit 14 to Visit20; Dosing Interval 4 = Visit 20 to Visit 26; Total = (Visit 2 throughVisit 26. ^(b)Day 26 of the previous dosing interval for DosingIntervals 2, 3, and 4. ^(c)Time to reach maximum mean TmP/GFR relativeto Day 0 of each dosing interval.

During each of the 4 dosing intervals, mean TmP/GFR increased to amaximum level by Day 7 and then declined prior to the next dosing; boththe pre-dose and postdose levels increased as the number of dosesincreased during the 120-day treatment period. As the doses increasedfrom the first to the fourth dosing interval, the maximum mean TmP/GFRincreased from 1.93±0.45 mg/dL to 2.97±0.67 mg/dL, the mean TmP/GFRprior to each dosing increased from 1.60±0.36 to 2.05±0.34 mg/dL, andmean AUCn increased from 5.23±6.12 mg·day/dL to 28.55±13.40 mg·day/dL.

Scatter plots of AUC_(last) and AUC_(n) for serum phosphorus versusAUC_(last) for TmP/GFR are presented in FIGS. 3A and 3B. Serumphosphorus and TmP/GFR were linearly correlated (Pearsoncorrelation=0.828 and 0.900, respectively).

The AUC_(last) for serum phosphorus change from Baseline (63.59±31.58mg·day/dL) and AUC_(last) for the TmP/GFR change from Baseline(68.42±37.96 mg·day/dL) were similar.

The mean calculated TmP/GFR values using a literature-reported equationversus mean TmP/GFR values manually read from the nomogram are shown inFIG. 4. The high correlation coefficient (Pearson correlation=0.9963)shows that the use of TmP/GFR calculated is comparable to TmP/GFRmanually read from the nomogram. Therefore, calculated TmP/GFR valuesare presented in this report. For 1 subject treated with placebo in thebone substudy, TmP/GRF levels remained essentially unchanged fromBaseline (1.3 mg/dL) to the end of the study (Visit 26) (1.6 mg/dL) withpostdose levels ranging from 1.3 to 1.8 mg/dL.

1,25-dihydroxyvitamin D

Mean serum 1,25(OH)2D levels for subjects treated with KRN23 aredisplayed graphically in FIG. 5.

During each of the 4 dosing intervals, mean serum 1,25(OH)2D levelsincreased to a maximum level by Day 3 or Day 7 and then returned to nearthe baseline level prior to the next dosing; both the pre-dose andpostdose levels increased as the number of doses increased during the120-day treatment period. As the doses increased from the first to thefourth dosing interval, the maximum mean serum 1,25(OH)2D levelsincreased from 53.1±25.44 to 94.1±45.99 pg/mL, the mean serum 1,25(OH)2Dlevels prior to each dose increased from 36.6±14.25 to 43.8±13.72 pg/mL,and the mean AUC_(n) increased from 144.81±209.12 to 1015.61±483.10pg·day/mL, see Table 8:

TABLE 8 Mean (±SD) 1,25(OH)₂D Levels and AUC of 1,25(OH)₂D Change fromBaseline in Subjects Treated with KRN23 (Efficacy Analysis Set)1,25(OH)₂D Levels AUC_(n) or AUC_(last) Time to of 1,25(OH)₂D reachMaximum Level Changefrom Dosing Day 0 (Predose) Maximum Mean (SD),Baseline Mean (SD), Interval^(a) Mean (SD), pg/mL Level^(b) pg/mL (pg ·day/mL) 1 36.6 (14.25), n = 24 Day 3 53.1 (25.44), n = 27  144.81(209.12), n = 24 2 37.1 (16.08), n = 24 Day 3 73.8 (32.24), n = 27 448.25 (244.32), n = 24 3 35.6 (15.55), n = 26 Day 7 91.9 (47.12), n =27  843.15 (428.67), n = 24 4 43.8 (13.72), n = 23 Day 3 94.1 (45.99), n= 26 1015.61 (483.10), n = 23 Total NA NA NA 2441.98 (1130.66), n = 241,25(OH)₂D = 1,25-dihydroxyvitamin D; AUC_(n) = area under theconcentration-time curve for the change from Baseline in serumphosphorus concentration during the nth dosing interval (n = 1, 2, 3, or4); AUC_(last) = AUC for the change for Baseline in serum phosphorusconcentration from Day 0 to Day 120 (or early withdrawal); NA = notapplicable; SD = standard deviation. ^(a)Dosing Interval 1 = Visit 2 toVisit 8; Dosing Interval 2 = Visit 8 to Visit 14; Dosing Interval 3 =Visit 14 to Visit 20; Dosing Interval 4 = Visit 20 to Visit 26; Total =Visit 2 to Visit 26. ^(b)Time to reach maximum mean 1,25(OH)₂D levelrelative to Day 0 of each dosing interval.

For 1 subject treated with placebo in the bone substudy, 1,25(OH)2Dlevels remained essentially unchanged from Baseline (67 pg/mL) to theend of the study (Visit 26) (53.0 pg/mL) with postdose levels rangingfrom 46 to 70 pg/mL.

Other Pharmacodynamic Results

The mean values in serum intact FGF23 levels at baseline are summarizedin table 9 below. A scatter plot of individual phosphorus concentrationversus intact FGF23 levels at baseline produced and no correlationbetween these 2 baseline parameters was observed (data not shown).

TABLE 9 Summary of Total Intact FGF23 by Visit/Day Efficacy Analysis SetTotal Intact FGF23 (pg/mL) Change (pg/mL) from Baseline Bone SubstudyBone Substudy Open Label Open Label Visit (Day)/ Treatment TotalTreatment Total Relative Day KRN23 KRN23 Placebo KRN23 KRN23 KRN23Placebo KRN23 Visit 2 (D0)/0 N 19 0 0 19 Mean 120.16 120.16 Median 92.4092.40 Std. Dev. 93.921 93.921 Range (Min-Max) (35.3, (35.3, 344.0)344.0) Visit 7 (D26)/26 N 26 1 0 27 19 0 0 19 Mean 10130.38 9440.0010104.81 9716.68 9716.68 Median 7950.00 9440.00 8060.00 8041.90 8041.90Std. Dev. 7212.678 NA 7073.861 5518.061 5518.061 Range (Min-Max)(2380.0, (9440.0, (2380.0, (2287.6, (2287.6, 35900.0) 9440.0) 35900.0)22081.0) 22081.0) Visit 13 (D54)/26 N 26 1 1 27 19 0 0 19 Mean 30757.3123600.00 154.00 30492.22 28931.95 28931.95 Median 22150.00 23600.00154.00 22200.00 21940.00 21940.00 Std. Dev. 24415.669 NA NA 23981.12418107.723 18107.723 Range (Min-Max) (5790.0, (23600.0, (154.0, (5790.0,(5748.9, 5748.9, 121000.0) 23600.0) 154.0) 121000.0) 70146.0) 70146.0)Visit 19 (D82)/26 N 26 1 1 27 19 0 0 19 Mean 70784.23 82100.00 40.0071203.33 64200.37 64200.37 Median 63350.00 82100.00 40.00 68200.0068131.90 68131.90 Std. Dev. 56631.443 NA NA 55574.383 33563.92033563.920 Range (Min-Max) (9990.0, (82100.0, (40.0, (9990.0, (9930.9,(9930.9, 305000.0) 82100.0) 40.0) 305000.0) 121890.0) 121890.0) Visit 25(D110)/26 N 25 1 1 26 18 0 0 18 Mean 126924.00 10500.00 37.60 122446.15124404.16 124404.16 Median 113000.00 10500.00 37.60 106500.00 116915.90116915.90 Std. Dev. 77668.008 NA NA 79450.333 61054.947 61054.947 Range(Min-Max) (22100.0, (10500.0, (37.6, (10500.0, (22040.9, (22040.9,391000.0) 10500.0) 37.6) 391000.0) 247681.0) 247681.0) End of Study -Visit N 25 1 1 26 18 0 0 18 Mean 105234.40 78200.00 36.00 104194.62112746.38 112746.38 Median 96400.00 78200.00 36.00 94800.00 106915.90106915.90 Std. Dev. 66001.220 NA NA 64884.702 69965.725 69965.725 Range(Min-Max) (3860.0, (78200.0, (36.0, (3860.0, (3808.4, (3808.4, 259000.0)78200.0) 36.0) 259000.0) 258890.0) 258890.0) Early Withdrawal N 1 0 0 11 0 0 1 Mean 3190.00 3190.00 3132.20 3132.20 Median 3190.00 3190.003132.20 3132.20 Std. Dev. NA NA NA NA Range (Min-Max) (3190.0, (3190.0,(3132.2, (3132.2, 3190.0) 3190.0) 3132.2) 3132.2)

Mean serum total and unbound FGF23 levels over time for subjects treatedwith KRN23 are summarized in Tables 10 and 11 below, respectively.

Pre-dose concentrations of total FGF23 increased with the number ofKRN23 doses and reached maximum levels after the fourth dosing interval(Day 110, 26 days after the last dose). Thereafter, a decreasing trendwas observed by the end of the study (Day 120, 36 days after the lastdose) (FIG. 6). Total FGF23 was 120.2±93.9 pg/mL at Baseline andincreased to 122446±79450 pg/mL on Day 110. Pre-dose concentration ofunbound FGF23 followed a similar pattern as total FGF23 and appearednegligible compared to total FGF23 when plotted in the same scale (FIG.6). Unbound FGF23 was 100±58 pg/mL at Baseline and increased to7024±5035 pg/mL on Day 110. Although unbound FGF23 increased after KRN23dosing, it was only a small fraction of the total FGF23 (5.74% on Day110).

As expected for the 1 subject in the placebo group, unbound FGF23 stayedessentially from predose (62.7 pg/mL) to the end of the study (59.8pg/mL) and ranged from 59.8 to 90.5 pg/mL.

A scatter plot of AUC_(last) for serum phosphorus change from Baselineversus intact FGF23 at Baseline is produced and there was no correlationbetween the AUC_(last) for serum phosphorus and baseline intact FGF23(Pearson correlation=0.0943).

No trends were noted for mean values over time for subjects treated withKRN23 for the following PD parameters: 25(OH)D, total calcium), ionizedcalcium, calcitonin, and iPTH in serum, or for the other PD parametersin urine, including 2-hour urine TRP, 2-hour urine calcium/creatinineratio, 2-hour urine FECa, 24-hour urine phosphorus, 24-hour urinecalcium, 24-hour urine creatinine, 24-hour urine calcium/creatinineratio.

Mean estradiol, testosterone, free testosterone, and SHBG over time wereobserved. No trends were noted for mean values over time in theseparameters.

TABLE 10 Summary of Total Intact FGF23 by Visit/Day Efficacy AnalysisSet Total Intact FGF23 (pg/mL) Change (pg/mL) from Baseline BoneSubstudy Bone Substudy Open Label Open Label Visit (Day)/ TreatmentTotal Treatment Total Relative Day KRN23 KRN23 Placebo KRN23 KRN23 KRN23Placebo KRN23 Visit 2 (D0)/0 N 19 0 0 19 Mean 120.16 120.16 Median 92.4092.40 Std. Dev. 93.921 93.921 Range (Min-Max) (35.3, (35.3, 344.0)344.0) Visit 7 (D26)/26 N 26 1 0 27 19 0 0 19 Mean 10130.38 9440.0010104.81 9716.68 9716.68 Median 7950.00 9440.00 8060.00 8041.90 8041.90Std. Dev. 7212.678 NA 7073.861 5518.061 5518.061 Range (Min-Max)(2380.0, (9440.0, (2380.0, (2287.6, (2287.6, 35900.0) 9440.0) 35900.0)22081.0) 22081.0) Visit 13 (D54)/26 N 26 1 1 27 19 0 0 19 Mean 30757.3123600.00 154.00 30492.22 28931.95 28931.95 Median 22150.00 23600.00154.00 22200.00 21940.00 21940.00 Std. Dev. 24415.669 NA NA 23981.12418107.723 18107.723 Range (Min-Max) (5790.0, (23600.0, (154.0, (5790.0,(5748.9, 5748.9, 121000.0) 23600.0) 154.0) 121000.0) 70146.0) 70146.0)Visit 19 (D82)/26 N 26 1 1 27 19 0 0 19 Mean 70784.23 82100.00 40.0071203.33 64200.37 64200.37 Median 63350.00 82100.00 40.00 68200.0068131.90 68131.90 Std. Dev. 56631.443 NA NA 55574.383 33563.92033563.920 Range (Min-Max) (9990.0, (82100.0, (40.0, (9990.0, (9930.9,(9930.9, 305000.0) 82100.0) 40.0) 305000.0) 121890.0) 121890.0) Visit 25(D110)/26 N 25 1 1 26 18 0 0 18 Mean 126924.00 10500.00 37.60 122446.15124404.16 124404.16 Median 113000.00 10500.00 37.60 106500.00 116915.90116915.90 Std. Dev. 77668.008 NA NA 79450.333 61054.947 61054.947 Range(Min-Max) (22100.0, (10500.0, (37.6, (10500.0, (22040.9, (22040.9,391000.0) 10500.0) 37.6) 391000.0) 247681.0) 247681.0) End of Study -Visit 26 (D120) N 25 1 1 26 18 0 0 18 Mean 105234.40 78200.00 36.00104194.62 112746.38 112746.38 Median 96400.00 78200.00 36.00 94800.00106915.90 106915.90 Std. Dev. 66001.220 NA NA 64884.702 69965.72569965.725 Range (Min-Max) (3860.0, (78200.0, (36.0, (3860.0, (3808.4,(3808.4, 259000.0) 78200.0) 36.0) 259000.0) 258890.0) 258890.0) EarlyWithdrawal N 1 0 0 1 1 0 0 1 Mean 3190.00 3190.00 3132.20 3132.20 Median3190.00 3190.00 3132.20 3132.20 Std. Dev. NA NA NA NA Range (Min-Max)(3190.0, (3190.0, (3132.2, (3132.2, 3190.0) 3190.0) 3132.2) 3132.2)

TABLE 11 Summary of Unbound Intact FGF23 by Visit/Day Efficacy AnalysisSet Unbound Intact FGF23 (pg/mL) Change (pg/mL) from Baseline BoneSubstudy Bone Substudy Open Label Open Label Treatment Total TreatmentTotal Visit/Day KRN23 KRN23 Placebo KRN23 KRN23 KRN23 Placebo KRN23Visit 2 (D0)/0 N 25 1 1 26 Mean 102.3 53.6 62.7 100.4 Median 82.1 53.662.7 81.9 Std. Dev. 58.06 NA NA 57.68 Range (Min-Max) (46, (54, (63,(46, 268) 54) 63) 268) Visit 7 (D26)/26 N 26 1 1 27 25 1 1 26 Mean 589.1935.0 77.8 601.9 359.2 881.4 15.1 379.3 Median 463.5 935.0 77.8 467.0391.5 881.4 15.1 392.3 Std. Dev. 676.23 NA NA 666.43 160.99 NA NA 188.07Range (Min-Max) (134, (935, (78, (134, (62, (881, (15, (62, 3780) 935)78) 3780) 724) 881) 15) 881) Visit 13 (D54)/26 N 26 1 1 27 25 1 1 26Mean 1347.5 11500.0 75.2 1723.5 1050.3 11446.4 12.5 1450.2 Median 1075.011500.0 75.2 1110.0 966.6 11446.4 12.5 1005.8 Std. Dev. 1171.18 NA NA2266.37 608.35 NA NA 2124.18 Range (Min-Max) (345, (11500, (75, (345,(284, (11446, (13, (284, 6220) 11500) 75) 11500) 2624) 11446) 13) 11446)Visit 19 (D82)/26 N 26 1 1 27 25 1 1 26 Mean 4083.3 36200.0 72.6 5272.83136.4 36146.4 9.9 4406.0 Median 3235.0 36200.0 72.6 3290.0 3114.936146.4 9.9 3172.1 Std. Dev. 4577.20 NA NA 7638.58 1554.23 NA NA 6650.49Range (Min-Max) (456, (36200, (73, (456, (364, (36146, (10, (364, 25200)36200) 73) 36200) 6574) 36146) 10) 36146) Visit 25 (D110)/26 N 25 1 1 2624 1 1 25 Mean 6812.8 12300.0 90.5 7023.8 5910.2 12246.4 27.8 6163.7Median 5860.0 12300.0 90.5 5920.0 5518.4 12246.4 27.8 5786.6 Std. Dev.5020.43 NA NA 5035.33 3080.70 NA NA 3271.26 Range (Min-Max) (1460,(12300, (91, (1460, (1368, (12246, (28, 1368, 26000) 12300) 91) 26000)12767) 12246) 28) 12767) End of Study - Visit 26 (D120) N 25 1 1 26 24 11 2 Mean 5834.4 3470.0 59.8 5743.5 5361.9 3416.4 −2.9 5284.1 Median6160.0 3470.0 59.8 5790.0 5713.0 3416.4 −2.9 5339.4 Std. Dev. 4043.21 NANA 3988.56 3637.57 NA NA 3582.18 Range (Min-Max) (252, (3470, (60, (252,(184, (3416, (−3, (184, 16000) 3470) 60) 16000) 15744) 3416) −3) 15744)Early Withdrawal N 1 0 0 1 1 0 0 1 Mean 213.0 213.0 130.9 130.9 Median213.0 213.0 130.9 130.9 Std. Dev. NA NA NA NA Range (Min-Max) (213, 213)(213, 213) (131, 131) (131, 131)Bone Formation Parameters

Mean serum BALP, P1NP, and osteocalcin values over time were alsoobserved. Mean P1NP levels increased from 64.1±30.7 ng/mL on Day 0 to123.0±75.1 ng/mL at the end of the study (Day 120). Numerical increaseswere also noted in serum osteocalcin (from 29.3±17.7 ng/mL on Day 0 to42.3±25.7 ng/mL the end of the study, Day 120) and BALP (from 28.3±12.8mcg/L on Day 0 to 38.1±23.3 mcg/L at the end of the study, Day 120).

For 1 subject treated with placebo in the bone substudy, mean values forserum BALP and P1NP remained essentially unchanged over time (BALP was20.7 mcg/L on Day 0 and 20.6 mcg/L at the end of the study, Day 120; andosteocalcin was 17.1 ng/mL on Day 0 and 19.4 ng/mL at the end of thestudy, Day 120). Numerical decreases were noted in mean serum P1NPlevels (from 91.0 ng/mL on Day 0 to 60.0 ng/mL at the end of the study,Day 120).

Bone Resorption Parameters

Mean serum CTx and NTx/creatinine ratio values over time were observed.Numerical increases were noted in serum CTx (from 752.0±389.5 pg/mL onDay 0 to 947.3±504.7 pg/mL at the end of the study, Day 120) andNTx/creatinine ratio (from 41.8±20.3 nmoL·BCE/mmoL on Day 0 to 56.9±34.0nmoL·BCE/mmoL at the end of the study, Day 120).

For 1 subject treated with placebo in the bone substudy, CTx remainedessentially unchanged over time (613.0 pg/mL on Day 0 and 570.0 pg/mL atthe end of the study, Day 120). Numerical increases were noted inNTx/creatinine ratio (from 25.0 nmoL·BCE/mmoL on Day 0 to 40.0nmoL·BCE/mmoL at the end of the study, Day 120).

Pharmacokinetic Results

Non-Compartmental Analysis

A summary of the total doses administered at each dosing interval isprovided in Table 12 below.

TABLE 12 Study Drug Administration Total Dose (mg/kg) by Dosing Interval(Safety Analysis Set) Open-Label Bone Substudy Total KRN23 KRN23 PlaceboKRN23 Dose 1 N 27 1 1 28 Mean (SD) 0.050 (0) 0.050 (NA) 0 0.050 (0)Median 0.050 0.050 (NA) 0 0.050 Range (0.05, 0.05) (0.05, 0.05) (0, 0)(0.05, 0.05) Dose 2 N 26 1 1 27 Mean (SD) 0.10 (0.01) 0.10 (NA) 0 0.10(0.01) Median 0.10 0.10 0 0.10 Range (0.05, 0.10) (0.10, 0.10) (0,0)(0.05, 0.10) Dose 3 N 26 1 1 27 Mean (SD) 0.28 (0.06) 0.30 (NA) 0 0.28(0.06) Median 0.30 0.30 0 0.30 Range (0.05, 0.30) (0.30, 0.30) (0, 0)(0.05, 030) Dose 4 N 25 1 1 26 Mean (SD) 0.48 (0.16) 0.30 (NA) 0 0.48(0.16) Median 0.60 0.30 0 0.60 Range (0.10, 0.60) (0.30, 0.30) (0,0)(0.10, 0.60) SD = standard deviation.

The mean serum concentrations of KRN23 are displayed graphically inFIGS. 7A and 7B. A summary of PK parameters for KRN23 are provided inTable 13 and Table 14 below.

TABLE 13 Intra-Dose-Escalation (Starting Dose 0.05 mg/kg SC) of KRN23Every 28-days to Adult Subjects with XLH (Pharmacokinetic Analysis Set)Dosing t_(max,n) C_(max,n) C_(min,n) AUC_(n) Interval Statistic (day)(ng/mL) (ng/mL) (ng · day/mL) 1 N 27 27 27 27 Mean (SD) 8.50 (2.90) 386(145) 147 (53.4) 7260 (2630) Min, Max 2.98, 14.9 193, 688 60.7, 2532980, 13200 Median 7.94 374 139 6560 CV % 34 38 36 36 2 N 27 27 27 27Mean (SD) 7.04 (1.71) 947 (307) 364 (130) 17900 (5310) Min, Max 3.95,11.9 356, 1710 119,662 6500, 28600 Median 6.96 870 373 16900 CV % 24 3236 30 3 N 27 27 27 27 Mean (SD) 7.45 (3.83) 2490 (843) 1080 (587) 50900(18000) Min, Max 1.96, 19.9 421, 3950 104, 2560 9500, 98000 Median 6.952540 1080 48300 CV % 51 34 54 35 4 N 26 26 26 26 Mean (SD) 7.00 (3.22)4750 (1800) 1470 (827) 109000 (40600) Min, Max 2.87, 15.0 1030, 8110166, 2890 23800, 178000 Median 6.89 5030 1310 103000 CV % 46 38 56 37AUC_(n) = area under the serum concentration-time curve in n-th dosinginterval; C_(max,n) = maximum serum concentration during the dosinginterval; C_(min,n) = predosed observed serum concentration at the endof n-th dosing interval; CV % = coefficient of variation, percent; Max =maximum; Min = minimum; SD = standard deviation; t_(max,n) = time ofmaximum observed serum concentration during n-th dosing interval; XLH =X-linked hypophosphatemia.

TABLE 14 Summary of Pharmacokinetic Parameters for KRN23 FollowingIntra-Dose-Escalation (Starting Dose 0.05 mg/kg) SubcutaneousAdministrations of KRN23 every 28-days to Adult Subjects with XLH (Datafrom all 4 Dosing Intervals) (Pharmacokinetic Analysis Set) AUC_(last)AUC_(inf) t_(1/2) CL/F V_(z)/F Statistic (μg · hr/mL)^(a) (μg ·hr/mL)^(b) (day) (mL/hr/kg)^(c) (mL/kg)^(d) t_(last) N 27 27 27 27 27 27Mean 4340 5240 16.4 0.186 98.3 122 SD 1320 1880 5.83 0.0780 34.6 2.79Min 1340 1570 5.58 0.0835 51.2 116 Median 4520 5170 15.8 0.161 86.1 122Max 6450 8990 29.5 0.472 212 125 CV % 30 36 35 42 35 2 AUC_(last) = areaunder the serum concentration-time curve from time 0 to the time of lastmeasurable concentration over all the dosing interval (Day 120 or earlywithdrawal) determined using the linear trapezoidal rule; AUC_(inf) =area under the serum concentration-time curve from 0 to infinity overall the dosing interval determined using the linear trapezoidal rule;t_(1/2) = terminal elimination half-life after 4th dosing; CL/F =apparent serum clearance; t_(1/2) = terminal elimination half-life afterfourth dosing interval; V_(z)/F = apparent volume of distribution;t_(last) = the time of last measurable concentration over all the dosinginterval. ^(a): AUC_(inf) was calculated as AUC_(last) + C_(last)/λz_(n)where C_(last) is the last measurable concentration over all the dosinginterval and λz_(n) is the rate constant associated with eliminationphase. ^(b): t_(1/2) was calculated as ln 2λz_(n). ^(c): CL/F wascalculated as Total Dose over 120 days of treatment/AUC_(inf). ^(d):V_(z)/F was calculated as CL/F and t_(1/2).

The mean tmax values were similar across the 4 dosing intervals andranged from 7.00 to 8.50 days. The individual tmax values ranged from1.96 to 19.9 days across all dosing intervals. The mean Cmax, n, Cmin,n, and AUCn values increased proportionally with increases in doses inDosing Intervals 1 to 4. The inter-subject variability for AUClast was30%. The inter-subject variability from Cmax, n and Cmin, n ranged from32% to 38% and from 36% to 56%, respectively, across all 4 dosingintervals. The mean t½ value for KRN23 after last dose in DosingInterval 4 was 16.4±5.8 days (individual values ranged from 5.58 to 29.5days). The mean CL/F and Vz/F for KRN23 calculated for all 4 intervalswere 0.186±0.078 mL/hr/kg and 98.3±34.6 mL/kg, respectively.

Pharmacokinetic and Pharmacodynamic Relationships

PK-PD Correlation for Serum Phosphorus: The plots of AUC_(n) andAUC_(last) of serum phosphorus change from baseline versus KRN23 AUC_(n)and AUC_(last), respectively, are shown in FIGS. 8A and 8B. The AUC_(n)for serum phosphorus change from baseline increased linearly withincrease of KRN23 AUCn (r=0.612). The AUClast for serum phosphoruschange from baseline increased linearly with increase of KRN23 AUClast(r=0.680).

PK-PD Correlation for TmP/GFR: The plots of AUCn and AUClast of TmP/GFRchange from baseline versus KRN23 AUCn and AUClast, respectively, areshown in FIGS. 9A and 9B. The AUCn for TmP/GFR change from Baselineincreased linearly with increases in KRN23 AUCn (r=0.810). The AUClastfor serum phosphorus change from Baseline increased linearly withincreases in KRN23 AUClast (r=0.698).

PK-PD Correlation for 1,25-dihydroxy vitamin D: The plots of AUCn andAUClast of 1,25(OH)₂D change from Baseline versus KRN23 AUCn andAUClast, respectively, are shown in FIGS. 10A and 10B. The AUCn for1,25(OH)₂D change from baseline increased linearly with increase inKRN23 AUCn (r=0.417). The AUClast for 1,25(OH)₂D change from Baselineincreased linearly with increases in KRN23 AUClast (r=0.272).

PK-PD Correlations for Other PD Markers: Plots of individual AUC forother PD parameters versus KRN23 AUC showed no correlation; theseincluded serum measures of albumin, ionized calcium, corrected calcium,calcitonin, iPTH, creatinine, total calcium, 25(OH)D, NTx, estradiol,free testosterone, total testosterone, SHBG; 24-hour urine measures ofcalcium, calcium/creatinine ratio, creatinine, phosphate; and 2-hoururine measures of creatinine, random calcium, calcium/creatinine ratio,FECa; phosphorous, and TRP. These results suggest that multiple-doseadministration of KRN23 did not affect any of these PD parameters.

PK-PD Correlations for Bone Markers: Scatter plots of AUC for boneformation markers (BALP, P1NP, and osteocalcin) and bone resorptionmarkers (CTx and NTx/creatinine ratio) change from baseline versus KRN23AUC showed a positive trend of increasing following KRN23 treatment.Further details are provided in the PK-PD report.

The results of the population PK-PD analysis between the change fromBaseline in phosphorus concentration and KRN23 concentration aresummarized in the table 15 below and in FIG. 11.

TABLE 15 Results of Population PK-PD Models for the Relationship BetweenSerum Phosphorus Changes From Baseline and KRN23 Concentrations in AdultSubjects with XLH Treated with KRN23 (Pharmacokinetic Analysis Set)Model (MOF) Parameters Typical Values BSV (%) E_(max) Model EC₅₀ (ng/mL)2742 61.3% (C_(KRN23) · E_(max))/(EC₅₀ + E_(max) (mg/dL) 1.788 11.0%C_(KRN23)) (MOF = 236.876) Residual additive error (ng/mL) 0.278 LinearModel α 0.138  106% ΔPhos = α · C_(KRN23) + β β 2.22 × 10⁻⁴ 36.8% (MOF =273.075) Residual additive error (ng/mL) 0.277 α = Slope; β = intercept;BSV = between-subject variability; ΔPhos = serum phosphorus change fromBaseline; C_(KRN23) = KRN23 concentration; EC₅₀ = drug concentrationwhen 50% of E_(max) is reached; E_(max) = maximum effect; MOF = minimumvalue of objective function: PK = pharmacokinetic. PD = pharmacodynamic;

The PK-PD relationship of serum KRN23 concentration and the increase inserum phosphorus levels was described by an Emax model. The Emax modelappeared to be show a better than linear model as indicated by low MOF.The maximum effect increase in serum phosphorus level was 1.788 mg/dL(Emax). KRN23 concentration reaching 50% of maximum effect (EC50) was2742 ng/mL.

The summary of KRN23 doses in each dosing cycle and the total dosesadministered is provided in Table 16 below.

TABLE 16 Summary of Dose Data for KRN23 Following Intra-SubjectDose-Escalation of Subcutaneous Administrations of KRN23 every 28-daysto Adult Subjects with X-Linked Hypophosphatemia Dose 1 Dose 2 Dose 3Dose 4 Total Dose Statistic (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) N 2727 27 26 27 Mean 0.05 0.10 0.28 0.48 0.89 SD 0.00 0.01 0.06 0.16 0.22Min 0.05 0.05 0.05 0.10 0.25 Max 0.05 0.10 0.30 0.60 1.05 CV % 0 10 2134 25

The mean serum concentrations of KRN23 are illustrated in FIG. 7. Thesummary PK parameters for KRN23 are provided in Table 13 to Table 14.The summary PK parameters with unit conversion are provided in Table 17.

TABLE 17 Summary of Pharmacokinetic Parameters (with Unit Conversion)for KRN23 Following Intra-Subject Dose-Escalation of SubcutaneousAdministrations of KRN23 every 28-days to Adult Subjects with X-LinkedHypophosphatemia (Data from All 4 Dosing Cycles) AUC_(last) AUC_(inf)t_(1/2) CL/F V_(z)/F t_(last) Statistic (μg · hr/mL) (μg · hr/mL) (hr)(mL/hr/kg) (mL/kg) (hr) N 27 27 27 27 27 27 Mean 4340 5240 394 0.18698.3 2920 SD 1320 1880 140 0.0780 34.6 67.0 Min 1340 1570 134 0.083551.2 2780 Median 4520 5170 380 0.161 86.1 2930 Max 6450 8990 708 0.472212 3000 CV % 30 36 35 42 35 2 Note: AUC_(last) was calculated as AUCover 120 days; AUC_(inf) was calculated as AUC_(last) +C_(last)/λz; CL/Fwas calculated as Total Dose over 120 days/AUC_(inf); V_(z)/F wascalculated from CL/F and t_(1/2)

All 27 subjects received a starting KRN23 dose of 0.05 mg/kg duringDosing Cycle 1; 26 of the 27 subjects received 0.1 mg/kg dose in DosingCycle 2; and 25 of the 27 subjects received KRN23 dose of 0.3 mg/kg inDosing Cycle 3. During the Dosing Cycle 4, 16 of the 26 subjectsreceived 0.6 mg/kg KRN23 dose, 9 of the 26 subjects received 0.3 mg/kgdose, and 1 of 26 subjects received 0.1 mg/kg dose. The mean±SD KRN23doses administered were: 0.05±0.00 mg/kg, 0.10±0.01 mg/kg, 0.28±0.06mg/kg, and 0.48±0.16 mg/kg during Dosing Cycles 1, 2, 3, and 4,respectively. The mean±SD total dose over 4 Dosing Cycles was 0.89±0.22mg/kg.

The mean tmax values were similar across four dosing cycles and rangedfrom 7.00 to 8.50 days. The individual tmax values ranged from 1.96 to19.9 day in all dosing cycles. The mean Cmax, n, Cmin, n, and AUCnvalues increased proportionally with increase in doses in Dosing Cycles1 to 4. The inter-subject variability for AUClast was 30%. Theinter-subject variability from Cmax, n and Cmin, n ranged from 32% to38% and from 36% to 56%, respectively, across all 4 dosing cycles.

The mean±SD t½ value for KRN23 after last dose in Dosing Cycle 4 was16.4±5.83 days (individual values ranged from 5.58 to 29.5 days). Themean±SD CL/F and Vz/F for KRN23 calculated for all 4 cycles were4.48±1.87 mL/day/kg and 98.3±34.6 mL/kg, respectively.

Pharmacokinetic Conclusion:

-   -   PK Exposure to KRN23 (assessed by Cmax, n, Cmin, n, AUCn values)        increased with increasing doses in a dose proportional manner.    -   Mean terminal half-life of KRN23 was 16.4 days (range: 5.58 to        29.5 days).    -   Mean tmax values were similar across four dosing cycles and        ranged from 7.00 to 8.50 days (range: 1.96 to 19.9 days).

PK-PD Correlation Conclusion:

-   -   The AUCn or AUClast for the change from baseline in serum        phosphorus increased linearly with increasing KRN23 PK exposure        (AUC_(n) or AUC_(last)).    -   The AUC_(n) or AUC_(last) for the change from baseline in        TmP/GFR increased linearly with increasing KRN23 PK exposure        (AUC_(n) or AUC_(last)).    -   The AUC_(n) or AUC_(last) for the change from baseline in        1,25-dihydroxy Vitamin D increased linearly with increasing        KRN23 PK exposure (AUC_(n) or AUC_(last)).    -   No meaningful effect of KRN23 treatment was observed on AUC_(n)        or AUC_(last) for change from baseline for most of the PD        parameters (albumin, ionized calcium, corrected calcium,        calcitonin, intact PTH, creatinine, total calcium, 25-Hydroxy        Vitamin D, NTX, estradiol, free testosterone, total        testosterone, SHBG in serum; calcium, calcium/creatinine ratio;        creatinine, phosphate in 24-hr urine; creatinine, random        calcium, calcium/creatinine ratio, FECa, phosphorous, TRP in        2-hr urine).    -   Increases in bone alkaline phosphatase, CTX, NTX/creatinine        ratio, osteocalcin, P1NP were observed following KRN23 treatment        but correlation with KRN23 exposure was weak.

Example 2: Second Phase I/II Clinical Study

Study Design and Methodology: This is an extended Phase I/II open-label,single-arm, repeat-dose, multicenter long-term extension studyconsisting of eligible XLH subjects from the first Phase I/11 clinicalstudy descried above. The study design consists of 2 periods:On-Treatment and Follow-Up. At Baseline, subjects were evaluated foreligibility into this study. All eligible subjects enrolled into theKRN23 open-label portion of this study received a starting dose of KRN23based on their serum phosphorus levels from the end-of-study visit(Visit 26, Day 120) of the first Phase I/II study, guided by adose-escalation algorithm and safety evaluations. During theOn-Treatment period, each subject receives open-label KRN23 administeredSC once every 28 days (up to 12 doses). Subsequent intra-subjectdose-escalation is based on serum phosphorus levels, guided by thedose-escalation algorithm for the study and safety evaluations. Duringthe On-Treatment period, subjects undergo a total of 48 scheduledclinical evaluations, from Visits 1-48. At least 12 of these scheduledevaluations are required clinic visits, whereas the remaining 36 visitscan be conducted at the clinic and/or by home health professionals. Uponcompletion of the On-Treatment period, subjects undergo an end-of-studyclinic visit (Visit 49), and an additional home or clinic visit (Visit50) during the Follow-Up period. Subjects participate for a total ofapproximately 13.5-months: approximately 11-months (48 weeks/336 days)during the On-Treatment period and approximately 2.5 months during theFollow-Up period. Eligible subjects from the first Phase I/II bonesubstudy may have participated in the extension portion of thissubstudy. Subjects were to continue the same regimen received in thefirst Phase I/II study. The bone substudy was closed in August 2013 dueto poor accrual; the analyses originally proposed may not be possible.Safety evaluations for the study include treatment-emergent adverseevent (TEAE) monitoring, safety laboratory parameters, immunogenicity,and physical examination findings.

Subjects who satisfactory completed both studies entry criteria wereeligible. The study population included males and females at least 18years of age with a documented clinical diagnosis of XLH and an intactfibroblast growth factor 23 (FGF23) level >30 pg/mL, ratio of renaltubular maximum reabsorption rate of phosphate to glomerular filtrationrate (TmP/GFR)<2.0 mg/dL, estimated glomerular filtration rate (eGFR)≥60mL/min (using Cockcroft-Gault formula), and corrected serum calciumlevel <10.8 mg/dL (if serum albumin was <4.0 g/dL).

Number of Subjects (planned and analyzed): Up to 35 subjects wereplanned; 23 subjects were screened for enrollment (21 in the open-labelportion of the study; 2 in the bone substudy); all subjects satisfiedeligibility criteria and were enrolled in the study.

Test Product, Dose and Mode of Administration, Batch Number: Open-labelKRN23 and KRN23 treatment in the bone substudy: KRN23 for SC injection,administered by SC injection at 0.05, 0.10, 0.30, 0.60, or 1.0 mg/kg tothe abdomen once every 28 days. (Batch Numbers: YU008B, YU008E, YU008F,YU009A, YU009B, YU011A, and YU011B). Subjects may be de-escalated to0.03 mg/kg if necessary.

Comparative Agent, Dose, and Mode of Administration, Batch Number: Bonesubstudy: KRN23 Placebo for SC injection, administered by SC injectionto the abdomen once every 28 days. (Batch number: PYU090908C andPYU120413A).

Duration of Treatment: The duration of treatment in this study was up toapproximately 11 months (48 weeks/336 days) during the On-Treatmentperiod and 2.5 months in the Follow-Up period, for a total ofapproximately 13.5 months.

Endpoints:

Primary: The primary endpoints were designed to evaluate the safety andefficacy of repeat-doses of KRN23 SC administration via the assessmentof the:

-   -   Number and % of adverse events (AEs) by severity and        relationship to investigational product    -   Number of clinically significant changes in vital signs,        laboratory tests, physical examinations, electrocardiogram        (ECG), renal ultrasound and cardiac computed tomography        (CT)/Coronary Calcium Scoring (CCS) and Aortic Calcium Scoring        (ACS)    -   Number and % of subjects with post-dose serum phosphorus levels:        ≤2.5 mg/dL; >2.5 mg/dL but ≤3.5 mg/dL; >3.5 mg/dL but ≤4.5 mg/dL        and >4.5 mg/dL    -   Number of subjects who develop anti-KRN23 antibodies        Secondary: The secondary endpoints were designed to evaluate the        following parameters by treatment group; changes are calculated        from Baseline (Visit 2, Day 0 of first Phase I/II clinical        trial):    -   Change in serum phosphorus levels    -   Changes in calcium homeostasis: 1,25(OH)2D (1,25-dihydroxy        vitamin D), 25(OH)D (25-hydroxy vitamin D), total calcium,        ionized calcium, calcitonin, and iPTH (intact parathyroid        hormone)    -   Changes in the ratio TmP/GFR, tubular reabsorption of phosphate        (TRP), urine calcium/creatinine ratio, and fractional excretion        of calcium (FECa), as measured from 2-hour urine    -   Changes in urinary phosphate, calcium, creatinine, and urine        calcium/creatinine ratio, as measured from 24-hour urine    -   Changes in bone biomarkers: serum BALP (bone alkaline        phosphatase), P1NP (procollagen type 1 N-propeptide), CTx        (carboxy-terminal cross-linked telopeptide of type I collagen),        and osteocalcin, and urinary NTx (amino terminal cross-linked        telopeptide of type I collagen)    -   Changes in sex hormones: estradiol, testosterone, free        testosterone, and sex hormone binding globulin (SHBG)    -   Characterize the population PK of KRN23 from cumulative dosing        across both the first and second Phase I/II clinical trial        studies    -   Changes in the QoL assessments in SF-36v2 (Outcomes Study        36-item Short Form, Volume 2) and WOMAC (Western Ontario and        McMaster Osteoarthritis Index)

Summary of Results: This clinical study summary is based on preliminarydata available as of the 12 Aug. 2013 data cut-off date.

Exposure: A total of 22 subjects were treated with KRN23 (21 in theopen-label portion of the study and 1 in the bone substudy) and 1subject was treated with Placebo in the bone substudy. In the initialdosing interval (Dose Interval 1), the mean KRN23 dose was 0.541±0.2039mg/kg. In subsequent dosing intervals, mean KRN23 dose was increased andvaried in a narrow range (0.733±0.2817 mg/kg to 0.865±0.2618 mg/kg).Most subjects treated with KRN23 (19/22, 86.4%) had received all 12planned doses as of the data cut-off date; the 1 Placebo-treated subjecthad received 3 doses of Placebo as of the data cut-off date.

Study Population: Among the 22 subjects treated with KRN23, the medianage was 42.5 years (range: 20 to 67 years), the majority (13/22, 59.1%)were women, and nearly all (21/22, 95.5%) were white/Caucasian while 1(4.5%) was Black/African American/African Caribbean. The median heightwas 148.79 cm (range: 121.9 to 170.2 cm) and median weight was 75.30 kg(range: 51.3 to 124.3 kg).

Serum phosphorus and TmP/GFR levels at Baseline were below the lowerlimit of the normal reference range for all subjects. At Baseline, theoverall mean 1,25(OH)2D level was within the normal reference range.Baseline unbound intact FGF23 levels ranged from 54 to 268 pg/mL forKRN23-treated subjects.

Efficacy Results:

-   -   At the pre-treatment baseline, all subjects had serum phosphorus        levels <2.5 mg/dL. Following KRN23 administration, the time of        maximum PD effect (peak) was observed on Days 7 and 14. After        the first dose of KRN23, 77.1% (Day 7) and 81.8% (Day 14) of        subjects had increased serum phosphorus levels within the target        range of >2.5 to ≤3.5 mg/dL. This peak effect was relatively        consistent throughout the study ranging from 45.5% to 81.8%. The        percentage of subjects within the target range was generally        similar on Day 7 and 14. No serum phosphorus level >4.5 mg/dL        was reported for any subject at any time point in the study.

Pharmacodynamic Results:

-   -   During each of the 12 dosing intervals, mean serum phosphorus        concentrations for KRN23-treated subjects increased        substantially by Day 7 or 14 and then declined but remained        above baseline at the end of the dose interval. All subjects        experienced an increase from baseline at all visits. The mean        serum phosphorus concentration increased from 1.85±0.282 mg/dL        at the pre-treatment baseline to a maximum level of 2.87±0.392        mg/dL on Day 14 of Dose Interval 1, and the maximum level        remained within the range of 2.71±0.428 mg/dL to 2.96±0.468        mg/dL throughout the study. At day 25 post-dose, 33%-57% of        subjects were still within the target range (>2.5 to ≤3.5 mg/dL)        and at the end of the dose interval 23.8%-38.1% of subjects        remained within the target range (>2.5 to ≤3.5 mg/dL).    -   TmP/GFR is the proposed mechanism by which KRN23-mediated        inhibition of excess FGF23 increases phosphorus levels. TmP/GFR        results followed the same pattern as that observed for serum        phosphorus. The mean TmP/GFR was increased from a pre-treatment        baseline level of 1.564±0.3012 into a maximum range from        2.408±0.7205 mg/dL to 2.921±0.5990 mg/dL on Day 14 of each        dosing interval. This magnitude of increase in TmP/GFR was        clinically meaningful and correlates closely with the increases        in serum phosphorus.    -   During each of the 12 dosing intervals, mean serum 1,25(OH)2D        levels increased to a maximum level by Day 7 and then declined        to a level comparable to Baseline prior to the next dosing.    -   No trends were noted in mean values over time for other serum        and urine PD parameters after KRN23 dosing compared to Baseline.        Serum parameters included 25(OH)D, total calcium, ionized        calcium, calcitonin and iPTH. Urine markers included 2-hour        measures of TRP, calcium/creatinine ratio, and FECa, and 24-hour        measures of phosphorus, calcium, creatinine, and        calcium/creatinine ratio.    -   Biomarkers of bone formation and resorption may provide an        indication of treatment effect. P1NP increased from 69.6±29.86        ng/mL at baseline of the first Phase I/II clinical trial study        to a maximum of 170.4±100.79 ng/mL, which represents a percent        change increase of 148.1%. Increases were observed at all        post-baseline measurements. Mean serum osteocalcin and BALP also        increased but to a lesser extent throughout the study. Increases        from baseline in bone resorption parameters (Ctx and NTx) were        also observed.

PK and PK-PD Relationships: Population PK analyses will be included inthe final study report. Any PK-PD relationship analyses will be includedin the final study report.

Quality of Life: Analyses of data from QoL assessments of the subjectsduring the second Phase I/II clinical trial study were not available asof the date of this report and will be included in the final studyreport.

Safety Results:

-   -   KRN23 was well tolerated following SC administration of up to 12        doses in this subject population.    -   There were no deaths or life-threatening TEAEs reported in the        study. SAEs were reported for 3 subjects treated with KRN23        (cervical spinal stenosis, breast cancer, and hypertensive        crisis; each 1 subject). Two subjects treated with KRN23 were        discontinued from the study due to TEAEs possibly related to        KRN23: severe or debilitating restless legs syndrome (RLS) that        was considered a DLT in 1 subject, and moderate nephrolithiasis        not considered a DLT but still leading to discontinuation of the        other subject.    -   TEAEs were reported for most of the subjects (20 subjects,        90.9%) treated with KRN23 and the 1 subject treated with Placebo        during the study. In the KRN23-treated group, the most common        TEAEs (reported for at least 3 subjects) were injection site        reaction, sinusitis, and arthralgia (each 5 subjects, 22.7%);        abdominal discomfort, back pain, pain in extremity and dizziness        (each 4 subjects, 18.2%); and vertigo, fatigue, gastroenteritis        viral, nasopharyngitis, headache, and RLS (each 3 subjects,        13.6%).    -   Treatment-related TEAEs were reported for 14 subjects (63.6%)        treated with KRN23. Injection site reaction (5 subjects, 22.7%);        arthralgia and RLS (each 3 subjects, 13.6%); and injection site        pain (2 subjects, 9.1%) were the only TEAEs classified as        treatment-related reported for more than 1 KRN23-treated        subject. Severe or disabling TEAEs were reported for 5 subjects        (22.7%) treated with KRN23: breast cancer unlikely study        drug-related, RLS possibly study drug-related, myalgia and        hypertensive crisis not study drug-related, cervical spinal        stenosis not study drug-related, and post-traumatic pain and        pain in extremity not study drug-related.    -   There was no discernible pattern of clinically significant        laboratory abnormalities during the study. Four subjects had        laboratory abnormalities considered by the Investigator to be        TEAEs: moderate neutrophil count decreased and white blood cell        count decreased possibly related to study drug; mild blood        pressure decreased possibly related to study drug; mild blood        triglycerides increased not related to study drug; and mild        alanine aminotransferase increased and aspartate        aminotransferase increased unlikely related to study drug and        mild blood creatine phosphokinase increased possibly related to        study drug. These events were not considered serious or        clinically significant, did not require treatment, and did not        result in discontinuation of the subjects from the study.    -   No adverse cardiac effects of relevance were noted in cardiac CT        scans or ECGs. A separate independent cardiologist evaluation of        the ECGs showed no evidence or changes of LVH by ESTES criteria        when comparing pretreatment ECGs (in the first or the second        Phase I/II clinical trial study) to post-treatment ECGs in the        second Phase I/II clinical trial study.    -   No adverse renal effects were noted on renal ultrasound, and no        treatment-related changes in iPTH, serum and 24-hour urinary        calcium were noted. No clinical significant ectopic        mineralization changes were observed.    -   No changes were observed in patterns of other safety parameters        (vital signs, physical and neurological examination findings)        that were suggestive of a treatment-related adverse effect.    -   No subject developed anti-KRN23 antibodies after dosing or        developed hypersensitivity reactions.    -   There was no evidence of increased calcifications in any        extra-osseous tissue studied, including kidney.        Conclusions:

Data from this study to date are consistent with the model that KRN23blocks FGF23 action after SC administration, which leads to a sustainedincrease in serum phosphorus levels due to increased tubularreabsorption of phosphate (TmP/GFR). Increased 1,25(OH)₂D was alsoobserved, as expected, based on the inhibition of the excess of FGF23.Increases in bone formation and resorption markers were also observed.Taken together, changes in these biomarkers support the hypothesis thatKRN23 may provide clinical improvements in bone quality, reverse thechanges associated with rickets, and eventually improve physicaloutcomes such as the bowing of the legs, pain, and short staturecharacteristic of patients with XLH.

Available data from this study demonstrated that KRN23 administered SCevery 28 days, in most cases at 0.6 or 1.0 mg/kg, improves peak serumphosphorus levels to the 2.5-3.5 mg/dL range from a mean pre-treatmentbaseline level of 1.85 mg/dL. This improvement persists over a period of48 weeks (336 days) and 12 KRN23 doses.

The data have also demonstrated that KRN23 treatment improves TmP/GFR;the effect persists for nearly 28 days following each treatment and ismaintained for 48 weeks and 12 doses. This result is consistent with theconcept that KRN23 reverses the decrease in the sodium-phosphateco-transporter in the kidney, resulting in substantial increases in theefficiency of phosphate reabsorption in the renal tubules. The magnitudeof increase in TmP/GFR was clinically meaningful and correlates withserum phosphorus, indicating the likely mechanism by whichKRN23-mediated inhibition of excess FGF23 achieves the desired effect.

Data from this study have also shown that KRN23 treatment increasesserum 1,25(OH)₂D levels. This is consistent with the concept that KRN23reverses the decreased expression of the 1-alpha hydroxylase required toconvert serum 25 hydroxy Vitamin D, into the 1,25 dihydroxy form thatactively stimulates phosphate absorption in the intestine. Improvementsin biomarkers of bone formation and resorption suggest an increase inbone remodeling. These improvements plus the increase in serumphosphorus is expected to be the mechanism by which bone quality willimprove in patients with XLH.

KRN23 was well tolerated by adult XLH subjects over a period of 48 weekswith up to 12 doses of drug administered SC at up to 1.0 mg/kg. Nodeaths or life threatening TEAEs occurred. Treatment-related TEAEsincluded injection site reaction; arthralgia and RLS; and injection sitepain. SAEs (assessed as unlikely to be or not study drug related) werereported for 3 subjects. No discernible clinically significant trends oflab abnormalities suggestive of a treatment-related adverse effect werenoted.

Calcifications of the kidney were a frequent finding in subjects atBaseline before KRN23 treatment, likely due to prior phosphate therapy.Clinical data on KRN23 to date, including that in this study using renalultrasound and ultra-fast CT scans of the heart, have not identifiedsignificant, novel appearance or any increase in calcifications in these2 most susceptible organs. In addition, no increases in serum or urinarycalcium or PTH were observed, and the phosphorus levels never exceededthe upper limit of normal, therefore the major biochemical parametersusually associated with ectopic mineralization did not occur with KRN23administration over this long-term observational study.

In conclusion, KRN23 treatment was well tolerated and increased serumphosphorus, TmP/GFR, and serum 1,25(OH)₂D levels in each dosing intervalthroughout the 48 weeks of treatment. No off target effects wereobserved. These clinically meaningful increases in pharmacodynamic andbiochemical markers, and the favorable safety profile in adult subjectswith XLH, suggest a potential utility of KRN23 treatment for patientswith XLH.

Efficacy Evaluation

Disease Characteristics at Baseline

Baseline disease characteristics, including the levels of serumphosphorus, TmP/GFR, 1,25(OH)₂D, and unbound intact FGF23 for subjectsin the Efficacy Analysis Set, are presented in Table 18 below. Serumphosphorus and TmP/GFR levels at Baseline were below the lower limit ofthe normal reference range for all subjects. At Baseline, the overallmean 1,25(OH)₂D level was within the normal reference range (Kratz2012). Baseline unbound intact FGF23 levels ranged from 54 to 268 pg/mLfor KRN23-treated subjects.

TABLE 18 Table Baseline Disease Characteristics (Efficacy Analysis Set)Open-label Bone Substudy Total KRN23 KRN23 Placebo KRN23 N = 21 N = 1 N= 1 N = 22 Unbound intact FGF23 (pg/mL) n 20 1 1 21 Mean (SD) 106.0(63.32) 53.6 (NA) 62.7 (NA) 103.5 (62.77) Min, Max (56, 268) (54, 54)(63, 63) (54, 268) Serum phosphorus (mg/dL)^(b) n 21 1 1 22 Mean (SD)1.85 (0.287) 2.00 (NA) 1.60 (NA) 1.85 (0.282) Min, Max (1.2, 2.3) (2.0,2.0) (1.6, 1.6) (1.2, 2.3) TmP/GFR (mg/dL)^(b) n 21 1 1 22 Mean (SD)1.570 (0.3076) 1.450 (NA) 1.250 (NA) 1.564 (0.3012) Min, Max (0.85,2.03) (1.45, 1.45) (1.25, 1.25) (0.85, 2.03) Serum 1,25 (OH)₂D (pg/mL) n18 1 1 19 Mean (SD) 36.1 (12.93) 42.0 (NA) 67.0 (NA) 36.4 (12.64) Min,Max (10, 61) (42, 42) (67, 67) (10, 61) Serum total calcium (mg/dL) n 211 1 22 Mean (SD) 9.11 (0.407) 9.30 (NA) 9.30 (NA) 9.12 (0.399) Min, Max(8.5, 10.2) (9.3, 9.3) (9.3, 9.3) (8.5, 10.2) Serum iPTH (pg/mL) n 21 11 22 Mean (SD) 84.2 (35.07) 74.0 (NA) 77.0 (NA) 83.7 (34.29) Min, Max(40, 143) (74, 74) (77, 77) (40, 143) BALP (μg/L) n 21 1 1 22 Mean (SD)30.94 (12.583) 34.40 (NA) 20.70 (NA) 31.10 (12.302) Min, Max (13.2,52.4) (34.4, 34.4) (20.7, 20.7) (13.2, 52.4) 24-hr urine calcium (mg/24hr) n 21 1 1 22 Mean (SD) 100.5 (69.27) 30.0 (NA) 54.0 (NA) 97.3 (69.25)Min, Max (11, 253) (30, 30) (54, 54) (11, 253) 24-hr urine creatinine(g/24 hr) n 21 1 1 22 Mean (SD) 1.383 (0.6689) 1.230 (NA) 1.040 (NA)1.376 (0.6536) Min, Max (0.54, 3.01) (1.23, 1.23) (1.04, 1.04) (0.54,3.01) 2-hr calcium/creatinine ratio, (mg/g creatinine) n 21 1 1 22 Mean(SD) 53.1 (39.83) 7.0 (NA) 36.0 (NA) 51.0 (40.09) Min, Max (11, 192)(7,7) 36,36) (7, 192) 1,25(OH)₂D = 1,25-dihydroxy vitamin D; BALP = bonealkaline phosphatase; FGF23 = fibroblast growth factor 23; hr = hour;iPTH = intact parathyroid hormone; Max = maximum; Min = minimum; NA =not applicable; SD = standard deviation; TmP/GFR = ratio of renaltubular maximum reabsorption rate of phosphate to glomerular filtrationrate. a: Baseline value is from Visit 2, Day 0 of the first Phase I/IIclinical trial. b: Normal ranges: 2.5 to 4.3 mg/dL (Kratz 2012) forserum phosphorus and 2.5-4.3 mg/dL for TmP/GFR (Walton et al. 1975).Primary Efficacy—Serum Phosphorus Levels

At the pre-treatment baseline (Visit 2, Day 0 of the first Phase I/IIclinical trial study), all 22 subjects treated with KRN23 (100%) and the1 subject treated with Placebo had serum phosphorus levels <2.5 mg/dL.During each 28 day dosing interval, the maximum serum phosphorus levels(peak) were achieved on Day 7 or 14 (Table 19). On Day 7 and 14 afterthe first KRN23 treatment (dosing interval 1), the serum phosphoruslevels of a large majority of subjects (17-18 subjects, 77.3%-81.8%) hadincreased from <2.5 mg/dL into the target range of >2.5 to ≤3.5 mg/dL.This peak effect was relatively consistent throughout the study rangingfrom 45.5% to 81.8%. The percentage of subjects within the target rangewas generally similar on both Day 7 and 14 throughout the study period.Peak serum phosphorus levels on Day 7 or 14 were increased into the >3.5to ≤4.5 mg/dL range for 0 to 3 KRN23-treated subjects (0-13.6%)throughout the study, but no serum phosphorus level >4.5 mg/dL wasreported for any subject at any time point.

TABLE 19 Proportion of Subject Treated with KRN23 with Serum PhosphorusLevels by Categories (Efficacy Analysis Population) Number (%) ofSubjects Treated with KRN23, N = 22 Serum Phosphorus Levels (mg/dL) ≤2.5 >2.5 to ≤3.5 >3.5 to ≤4.5 >4.5 Baseline 22 (100.0) 0 (0.0) 0 (0.0) 0(0.0) Dosing Interval 1 Visit 1/0 21 (95.5) 0 (0.0) 0 (0.0) 0 (0.0)Visit 2/7 4 (18.2) 17 (77.3) 1 (4.5) 0 (0.0) Visit 3/14 4 (18.2) 18(81.8) 0 (0.0) 0 (0.0) Visit 4/25 15 (68.2) 7 (31.8) 0 (0.0) 0 (0.0)Dosing Interval 2 Visit 5/0 15 (68.2) 6 (27.3) 0 (0.0) 0 (0.0) Visit 6/76 (27.3) 13 (59.1) 2 (9.1) 0 (0.0) Visit 7/14 4 (18.2) 15 (68.2) 2 (9.1)0 (0.0) Visit 8/25 7 (31.8) 13 (59.1) 0 (0.0) 0 (0.0) Dosing Interval 3Visit 9/0 12 (54.5) 9 (40.9) 0 (0.0) 0 (0.0) Visit 10/7 5 (22.7) 15(68.2) 1 (4.5) 0 (0.0) Visit 11/14 6 (27.3) 15 (68.2) 0 (0.0) 0 (0.0)Visit 12/25 10 (45.5) 11 (50.0) 0 (0.0) 0 (0.0) Dosing Interval 4 Visit13/0 13 (59.1) 8 (36.4) 0 (0.0) 0 (0.0) Visit 14/7 7 (31.8) 11 (50.0) 3(13.6) 0 (0.0) Visit 15/14 3 (13.6) 17 (77.3) 1 (4.5) 0 (0.0) Visit16/25 10 (45.5) 11 (50.0) 0 (0.0) 0 (0.0) Dosing Interval 5 Visit 17/013 (59.1) 7 (31.8) 0 (0.0) 0 (0.0) Visit 18/7 5 (22.7) 15 (68.2) 0 (0.0)0 (0.0) Visit 19/14 8 (36.4) 13 (59.1) 0 (0.0) 0 (0.0) Visit 20/25 9(40.9) 12 (54.5) 0 (0.0) 0 (0.0) Dosing Interval 6 Visit 21/0 15 (68.2)5 (22.7) 0 (0.0) 0 (0.0) Visit 22/7 3 (13.6) 15 (68.2) 2 (9.1) 0 (0.0)Visit 23/14 3 (13.6) 15 (68.2) 1 (4.5) 0 (0.0) Visit 24/25 12 (54.5) 8(36.4) 0 (0.0) 0 (0.0) Dosing Interval 7 Visit 25/0 13 (59.1) 7 (31.8) 0(0.0) 0 (0.0) Visit 26/7 4 (18.2) 16 (72.7) 0 (0.0) 0 (0.0) Visit 27/145 (22.7) 14 (63.6) 0 (0.0) 0 (0.0) Visit 28/25 10 (45.5) 10 (45.5) 0(0.0) 0 (0.0) Dosing Interval 8 Visit 29/0 13 (59.1) 6 (27.3) 0 (0.0) 0(0.0) Visit 30/7 7 (31.8) 8 (36.4) 3 (13.6) 0 (0.0) Visit 31/14 7 (31.8)11 (50.0) 0 (0.0) 0 (0.0) Visit 32/25 13 (59.1) 6 (27.3) 0 (0.0) 0 (0.0)Dosing Interval 9 Visit 33/0 11 (50.0) 7 (31.8) 0 (0.0) 0 (0.0) Visit34/7 5 (22.7) 12 (54.5) 1 (4.5) 0 (0.0) Visit 35/14 5 (22.7) 12 (54.5) 0(0.0) 0 (0.0) Visit 36/25 11 (50.0) 7 (31.8) 1 (4.5) 0 (0.0) DosingInterval 10 Visit 37/0 12 (54.5) 7 (31.8) 0 (0.0) 0 (0.0) Visit 38/7 7(31.8) 10 (45.5) 2 (9.1) 0 (0.0) Visit 39/14 8 (36.4) 9 (40.9) 2 (9.1) 0(0.0) Visit 40/25 10 (45.5) 9 (40.9) 0 (0.0) 0 (0.0) Dosing Interval 11Visit 41/0 14 (63.6) 5 (22.7) 0 (0.0) 0 (0.0) Visit 42/7 5 (22.7) 13(59.1) 1 (4.5) 0 (0.0) Visit 43/14 8 (36.4) 10 (45.5) 1 (4.5) 0 (0.0)Visit 44/25 9 (40.9) 10 (45.5) 0 (0.0) 0 (0.0) Dosing Interval 12 Visit45/0 14 (63.6) 5 (22.7) 0 (0.0) 0 (0.0) Visit 46/7 8 (36.4) 9 (40.9) 2(9.1) 0 (0.0) Visit 47/14 9 (40.9) 10 (45.5) 0 (0.0) 0 (0.0) Visit 48/2512 (54.5) 7 (31.8) 0 (0.0) 0 (0.0)

Note that each row of data presents available data for that visit/timepoint, and the total number of subjects (and associated percentages) ateach visit may not add up to 22 (or 100%), where there is missing data.The percentage of subjects whose serum phosphorus remained within thetarget range (>2.5 to ≤3.5 mg/dL) at each time point was calculated witha denominator based on the total of 22 subjects who had received anynumber of KRN23 doses, whether or not serum phosphorus data wereobtained for all of these subjects at that time point. As of the datacut-off date, 3 subjects had not received all of their planned KRN23doses (1 subject received 1 dose and was discontinued; 1 subject hadreceived 5 doses and 1 subject had received 7 doses and remained onstudy), and at some time points serum phosphorus data were not availablefor all subjects who had been treated and remained on study. Therefore,in some instances, the calculated percentage may underestimate theresponder rate of subjects achieving a serum phosphorus > than 2.5mg/dL.

For the 1 subject treated with Placebo in the bone substudy, serumphosphorus levels remained essentially unchanged from Baseline (1.60mg/dL), with post-dose levels ranging from 1.70 to 2.00 mg/dL.

In summary, KRN23 increased serum phosphorus levels from baseline in allsubjects at almost all data points either at the peak or through, andmost treated subjects achieved the target therapy goal (>2.5 to ≤3.5mg/dL). This effect was maintained for up to 48 weeks.

Pharmacodynamic Results

Serum Phosphorus: Mean serum phosphorus levels are displayed graphicallyin FIG. 12.

Since the dose was adjusted based on the peak and trough phosphoruslevels and on the pre-defined adjustment regimen, the KRN23 dose variesfrom subject to subject and throughout the course of the study. The meanKRN23 dose ranged from 0.541±0.2039 mg/kg to 0.865±0.2618 mg/kg in thestudy. During each of the 12 dosing intervals, mean serum phosphorusconcentrations increased to clinically meaningful maximum levels by Day7 or 14 and then declined but did not return to the pre-dose level priorto the next dosing. All subjects experienced an increase from baselineat all visits. The mean serum phosphorus concentration of these subjectsincreased from 1.85±0.282 mg/dL at pre-treatment Baseline (the firstPhase I/11 clinical trial study Day 0) to a maximum level of 2.87±0.392mg/dL on Day 14 of Dose Interval 1, and the maximum level remainedwithin the range of 2.71±0.428 mg/dL to 2.96±0.468 mg/dL throughout thestudy. The maximum mean change from Baseline in serum phosphorusremained within the range from 0.87±0.444 mg/dL to 1.10±0.412 mg/dLthroughout the study (Table 20). The mean serum phosphorus concentrationat the end of the dose interval (Day 0) remained above baseline and therange was from 2.25±0.353 mg/dL to 2.52±0.436 mg/dL.

TABLE 20 Maximum Mean (±SD) Serum Phosphorus Levels and Maximum MeanChange from Baseline for Subjects Treated with KRN23 (Efficacy AnalysisSet) Serum Phosphorus Levels Maximum Change Maximum from Time to ReachLevel Baseline^(b) Dosing Day 0 Mean (SD), Maximum Mean (SD), Mean (SD),Interval mg/dL Level^(a) mg/dL mg/dL  1 1.88 (0.303), n = 21 Day 14, n =22 2.87 (0.392) 1.01 (0.375)  2 2.25 (0.353), n = 21 Day 14, n = 21 2.95(0.441) 1.09 (0.467)  3 2.43 (0.349), n = 21 Day 7, n = 21 2.96 (0.468)1.10 (0.412)  4 2.42 (0.396), n = 21 Day 7, n = 21 2.92 (0.560) 1.06(0.516)  5 2.35 (0.376), n = 20 Day 7, n = 20 2.82 (0.396) 0.95 (0.435) 6 2.35 (0.330), n = 20 Day 7, n = 20 2.94 (0.387) 1.09 (0.436)  7 2.37(0.385), n = 20 Day 7, n = 20 2.83 (0.315) 0.99 (0.427)  8 2.40 (0.422),n = 19 Day 7, n = 18 2.87 (0.542) 1.03 (0.532)  9 2.42 (0.403), n = 18Day 7, n = 18 2.79 (0.456) 0.94 (0.402) 10 2.52 (0.436), n = 19 Day 7, n= 19 2.85 (0.550) 1.01 (0.549) 11 2.29 (0.397), n = 19 Day 7, n = 192.71 (0.428) 0.87 (0.444) 12 2.25 (0.301), n = 19 Day 7, n = 19 2.73(0.427) 0.88 (0.468) AUC_(n) = area under the concentration-time curvefor the change from Baseline in serum phosphorus concentration duringthe nth dosing interval (n = 1, 2, 3, etc.); NA = not applicable; SD =standard deviation. ^(a)Time to reach maximum mean serum phosphoruslevel relative to Day 0 of each dosing interval. ^(b)Mean Baseline serumphosphorus level: 1.85 ± 0.282 mg/dL (n = 22). Maximum change fromBaseline was achieved on the same relative dosing day as the maximummean serum phosphorus level for all dosing intervals.

Peak and trough fluctuations from the maximum mean serum phosphorusvalue on Day 7 or 14 of a dosing interval to the minimum value on Day 0of the following dosing interval were low throughout the study. Forexample, the maximum mean peak value of 2.96±0.468 mg/dL (Visit 10, DoseInterval 3, Day 7) was followed by a trough value of 2.42±0.396 mg/dL(Visit 13, Dose Interval 4, Day 0), for a 0.54 mg/dL difference (22.3%).The minimum mean trough values of 2.25±0.353 mg/dL (Visit 5, DoseInterval 2, Day 0) and 2.25±0.301 (Visit 45, Dose Interval 12, Day 0)followed peak values in the previous dosing intervals of 2.87±0.392mg/dL (Visit 3, Dose Interval 1, Day 14) and 2.71±0.428 mg/dL (Visit 42,Dose Interval 11, Day 7), for differences of 0.62 mg/dL (27.6%) and 0.46mg/dL (20.4%), respectively. The differences for 10 of the 11 of theavailable peak-trough pairs fell within the range of 17.9% to 27.6%;displaying a consistent PD effect over time. Inter-subject variabilitywas also relatively low for serum phosphorus concentration. For example,between-subject variation of post-dose serum phosphorus levels(SD/mean×100%) was in the range of 13.4% to 15.8% for both paired troughand peak concentrations at the beginning of the study (Dose Interval1-2) and the end of the study (Dose Interval 11-12). These resultshighlight the minimal variability and consistent PD effect.

Renal Tubular Maximum Reabsorption Rate of Phosphate/GlomerularFiltration Rate

Two hour urine samples collected on Days 14 and 25 after each KRN23 dosein each dosing interval were used to calculate TmP/GFR. Mean TmP/GFRvalues are displayed graphically in FIG. 13.

TmP/GFR is the proposed mechanism by which KRN23-mediated inhibition ofexcess FGF23 increases phosphorus levels. TmP/GFR followed the samepattern as serum phosphorus, suggesting that the increased serumphosphorus level following KRN23 treatment is mainly due to increasedphosphorus reabsorption from renal proximal tubules. In all dosingintervals and all subjects, mean TmP/GFR was increased by a clinicallysignificant amount on Day 14 compared to Baseline (Visit 2, Day 0 ofthis study), and then declined toward the pre-dose level prior to thenext dosing (Table 23). Mean TmP/GFR was increased from a Baseline levelof 1.564±0.3012 to a level within the range from 2.408±0.7205 mg/dL to2.921±0.5990 mg/dL on Day 14 of each dosing interval; the Day 14 changefrom Baseline ranged from 0.849±0.7665 to 1.352±0.5892. Day 25 TmP/GFRlevels ranged from 2.195±0.3819 mg/dL to 2.551±0.6538 mg/dL, and thechange from Baseline ranged from 0.636±0.3085 to 1.004±0.6777.

TABLE 23 Mean (±SD) TmP/GFR Levels and Change from Baseline in SubjectsTreated with KRN23 (Efficacy Analysis Set) TmP/GFR Levels Change fromBaseline^(a) to Day 14 Value Change from Day 25 Value Day 25 Dosing Mean(SD), Baseline^(a) to Day 14 Mean (SD), Mean (SD), Interval mg/dL Mean(SD), mg/dL mg/dL mg/dL  1 2.655 (0.5660), n = 22 1.090 (0.4996) 2.401(0.5543), n = 19 0.823 (0.5708)  2 2.907 (0.7448), n = 21 1.336 (0.7208)2.551 (0.6538), n = 20 1.004 (0.6777)  3 2.602 (0.4876), n = 21 1.031(0.5076) 2.282 (0.4864), n = 21 0.711 (0.4812)  4 2.921 (0.5990), n = 201.352 (0.5892) 2.295 (0.4892), n = 21 0.724 (0.5208)  5 2.600 (0.6415),n = 21 1.030 (0.5816) 2.314 (0.4427), n = 20 0.754 (0.4601)  6 2.735(0.5993), n = 19 1.206 (0.6388) 2.401 (0.4807), n = 20 0.847 (0.5337)  72.754 (0.5730), n = 18 1.238 (0.5875) 2.422 (0.5060), n = 19 0.862(0.6200)  8 2.677 (0.6045), n = 18 1.144 (0.6069) 2.392 (0.5948), n = 190.832 (0.5492)  9 2.636 (0.5144), n = 17 1.116 (0.4958) 2.272 (0.4700),n = 18 0.739 (0.4822) 10 2.408 (0.7205), n = 19 0.849 (0.7665) 2.234(0.6327), n = 19 0.674 (0.7502) 11 2.595 (0.6046), n = 19 1.036 (0.5812)2.292 (0.5204), n = 19 0.733 (0.5231) 12 2.572 (0.5380), n = 19 1.013(0.5514) 2.195 (0.3819), n = 19 0.636 (0.3085) NA = not applicable; SD =standard deviation: Tmp/GFR = ratio of the renal tubular maximumreabsorption rate of phosphate to glomerular filtration rate.^(a)Baseline mean TmP/GFR: 1.564 ± 0.3012 mg/dL (n = 22).

For the 1 subject treated with Placebo in the bone substudy, TmP/GFRlevels remained little changed from Baseline (1.250 mg/dL) to the lastevaluation completed as of the data cut-off date (Visit 8, Dose Interval2, Day 25) (1.650 mg/dL) with post-dose levels ranging from 1.550 to2.000 mg/dL.

1,25-Dihydroxy Vitamin D

Mean serum 1,25(OH)₂D levels for subjects treated with KRN23 aredisplayed graphically in FIG. 14.

During each of the 12 dosing intervals, mean serum 1,25(OH)₂D levelsincreased to a maximum level by Day 7 and then declined to a levelcomparable to Baseline prior to the next dosing. There is a trend towarddecreased peak levels for 1,25(OH)₂D over time from the first to thelast dosing interval (Table 24). The mean serum 1,25(OH)₂D level atBaseline (Visit 2, Day 0 of the first Phase I/II clinical trial study)was 36.4±12.64 and was increased on Day 7 of each dosing interval to amaximum within the range from 53.1±20.28 pg/mL to 92.0±43.21 pg/mL. TheDay 7 maximum change from Baseline in serum 1,25(OH)₂D level remainedwithin the range from 19.6±15.91 pg/mL to 63.4±35.52 pg/mL throughoutthe study.

For the 1 subject treated with Placebo in the bone substudy, 1,25(OH)₂Dlevels remained little changed from Baseline (67.0 pg/mL) to the lastevaluation completed as of the data cut-off date (Visit 10, DoseInterval 3, Day 7) (50.0 pg/mL) with post-dose levels ranging from 34.0to 72.0 pg/mL.

TABLE 24 Maximum Mean (±SD) 1,25(OH)₂D Levels and Maximum Mean Changefrom Baseline in Subjects Treated with KRN23 (Efficacy Analysis Set)1,25(OH)₂D Levels Maximum Time to reach Level Maximum Dosing Day 0(Pre-dose) Maximum Mean (SD), Change from Interval Mean (SD), pg/mLLevel^(a) pg/mL Baseline  1 30.0 (12.79), n = 21 Day 7 92.0 (43.21), n =21 63.4 (35.52), n = 18  2 39.4 (15.85), n = 21 Day 7 86.3 (31.49), n =21 55.3 (25.16), n = 18  3 43.1 (16.39), n = 21 Day 7 77.9 (25.89), n =21 45.2 (20.72), n = 18  4 43.0 (17.06), n = 21 Day 7 72.2 (20.67), n =20 39.5 (19.02), n = 17  5 40.8 (13.61), n = 20 Day 7 69.4 (24.50), n =20 37.1 (20.90), n = 17  6 41.7 (20.43), n = 20 Day 7 66.2 (21.96) n =20 31.7 (17.05), n = 17  7 35.5 (15.50), n = 20 Day 7 61.5 (19.03), n =20 27.9 (15.60), n = 17  8 35.9 (13.73), n = 19 Day 7 56.5 (21.74), n =18 23.4 (15.36), n = 15  9 31.4 (10.41), n = 18 Day 7 59.2 (24.12), n =18 27.0 (22.29), n = 15 10 33.6 (11.49), n = 19 Day 7 57.9 (27.05), n =19 24.3 (24.30), n = 16 11 29.7 (10.54), n = 19 Day 7 53.1 (20.28), n =19 19.6 (15.91), n = 16 12 33.6 (10.85), n = 19 Day 7 57.3 (20.18), n =19 23.5 (15.43), n = 16 1,25(OH)₂D = 1,25-dihydroxy vitamin D; NA = notapplicable; SD = standard deviation. ^(a)Time to reach maximum mean1,25(OH)₂D level relative to Day 0 of each dosing interval.Other Pharmacodynamic Results

The mean values of serum total intact FGF23 levels at Baseline wererecorded. The total FGF23 ECLA assay measures endogenous FGF23 in thepresence of KRN23, whether complexed or unbound in serum. Standards(recombinant human FGF23 [rhFGF23]) and samples are incubated withexcess amounts of KRN23 in a buffer matrix then detected by the assay.The assay measures the KRN23/FGF23 complex, which represents allendogenous FGF23 in the sample. The assay is limited since theendogenous FGF23/KRN23 complex in serum does not yield a parallel linearresponse to the rhFGF23/KRN23 complex standard curve, hence all resultsfor total FGF23 using the ECLA are relative to the standard curve andcan only be used to describe trends of increases or decreases inendogenous FGF23 concentration.

For the subjects who were to be treated with KRN23 in the first PhaseI/II clinical trial, the mean serum total intact FGF23 level was94.7±105.19 pg/mL at Baseline (Visit 2, Day 0 in first Phase I/IIclinical trial) prior to any treatment with KRN23. After completingKRN23 treatments that study, this value had been increased to42100.0±31524.88 pg/mL at the time of the first visit in second PhaseI/II clinical trial (Visit 1, Dosing Interval 1, Day 0). Mean serumtotal intact FGF23 generally increased further with an increasing numberof KRN23 doses in second Phase I/II clinical trial and reached a maximumof 510818.8±354649.07 pg/mL at Visit 45 (Dose Interval 12, Day 0) (FIG.15).

The assay for unbound FGF23 is difficult to conduct in the presence oflarge amounts of bound FGF23, and it is difficult to be confident thatthe levels of free FGF23 are actually free in the circulation or aremerely dissociating in the assay. Using the assay as developed, the meanlevels of unbound intact FGF23 followed a pattern similar to that oftotal intact FGF23 through Visit 33 in subjects treated with KRN23; themean values of the unbound form then decreased at Visits 37, 41 and 45before rising again at Visit 49 (FIG. 16). The mean unbound intact FGF23level was 103.5±62.77 pg/mL at Baseline and increased to25283.3±33810.42 pg/mL at Visit 33 and 25657.9±13461.86 pg/mL at Visit49. Although unbound intact FGF23 increased after KRN23 dosing, itrepresented only a small fraction of the total intact FGF23; the meanpeak value of the unbound form is 5% that of total intact FGF23. It istherefore difficult to determine if this is accurate in this context.

KRN23 treatment led to increased serum phosphorus levels despite theapparent increases in total and unbound intact FGF23, which wouldsuggest that whether free or not, the apparent free FGF23 was not activein vivo in this context.

In contrast to the effect of KRN23 treatment on serum phosphorus andTmP/GFR, no trends were noted in mean values over time for subjectstreated with KRN23 for the following PD parameters: 25(OH)D, totalcalcium, ionized calcium, calcitonin, and iPTH in serum; or for theother PD parameters in urine, including 2-hour urine TRP, 2-hour urinecalcium/creatinine ratio, 2-hour urine FECa, 24-hour urine phosphorus,24-hour urine calcium, 24-hour urine creatinine, and 24-hour urinecalcium/creatinine ratio.

Mean estradiol, testosterone, free testosterone, and SHBG over time forsubjects treated with KRN23 were measured as well. No trends were notedfor mean values over time in these parameters.

Bone Biomarkers

Bone Formation Parameters: Biomarkers of bone formation and resorptionmay provide an indication of treatment effect. Mean serum BALP, P1NP,and osteocalcin values over time were measured. P1NP increased from69.6±29.86 ng/mL at baseline of the first Phase I/II clinical trialstudy, to a maximum of 170.4±100.79 ng/mL at Visit 17, which representsa percent change increase of 148.1%. The value remained elevated at155.2±75.95 ng/mL on Visit 33/Dose Interval 9/Day 0. Serum osteocalcinand BALP also increased but to a lesser extent throughout the study.These data suggest that KRN23 increases bone formation and this can bethe mechanism by which the bone quality may improve, the rickets changesreversed and the bone shape restored.

Bone Resorption Parameters: Mean serum CTx and NTx values over time weremeasured. For subjects treated with KRN23, numerical increases werenoted in serum CTx (from 845.1±345.77 pg/mL on Visit 1/Dose Interval1/Day 0 to 1090.5±570.55 pg/mL on Visit 17/Dose Interval 5/Day 0 and1157.2±542.25 pg/mL on Visit 33/Dose Interval 9/Day 0) These changes inCTX are in the order of approximately 73% change from baseline. NTx alsoincreased (from 246.2±578.65 nm bone collagen equivalents [BCE]/mL onVisit 1/Dose Interval 1/Day 0 to 346.5±611.50 nm BCE on

Visit 17/Dose Interval 5/Day 0 and 344.6±566.55 nm BCE on Visit 33/DoseInterval 9/Day 0). These data demonstrate that KRN23 increases boneresorption as well as bone formation, and this increase in boneremodeling and turnover plus the normalization of the serum phosphoruslevels is likely the key physiologic mechanism by which KRN23 willimprove bone quality.

Efficacy Conclusions

-   -   In this study serum phosphorus is considered the efficacy end        point and a key PD parameter. At the pre-treatment baseline, all        subjects had serum phosphorus levels <2.5 mg/dL. Following KRN23        administration, the time of maximum PD effect (peak) was        observed on Days 7 and 14. After the first dose of KRN23, 77.1%        (Day 7) and 81.8% (Day 14) of subjects had increased serum        phosphorus levels within the target range of >2.5 to ≤3.5 mg/dL.        This peak effect was relatively consistent throughout the study        ranging from 45.5% to 81.8%. The percentage of subjects within        the target range was generally similar on Day 7 and 14. No serum        phosphorus level >4.5 mg/dL was reported for any subject at any        time point in the study.

Pharmacodynamic

-   -   TmP/GFR is the proposed mechanism by which KRN23-mediated        inhibition of excess FGF23 increases phosphorus levels. KRN23        increased TmP/GFR; the magnitude of increase in TmP/GFR was        clinically meaningful and correlates closely with the increases        in serum phosphorus. The observed PD effect highlights the        likely mechanism of KRN23 action by increasing phosphorus        reabsorption and the distal tubular level serum phosphorus,        thereby achieving the desired clinical effect.    -   During each of the 12 dosing intervals, mean serum 1,25(OH)2D        levels increased to a maximum level by Day 7 and then declined        to a level comparable to Baseline prior to the next dosing. 1,25        vitamin D is another PD parameter intimately linked to the        mechanism of action of KRN23, and also increased following the        same temporal profile: rapid increase post-dose, peaking at day        7, and returning towards baseline levels at the end of the dose        interval.    -   All other metabolic parameters: 25(OH)D, total calcium, ionized        calcium, calcitonin and iPTH; urine measures of TRP,        calcium/creatinine ratio, and FECa, and 24-hour urine measures        of phosphorus, calcium, creatinine, and calcium/creatinine        ratio, did not change significantly. Therefore no off target        effects were observed; changes in on-target PD parameters,        essentially serum phosphorus and 1,25(OH)2D, did not appear to        alter calcium and PTH metabolism.    -   The markers of bone formation increased significantly,        particularly P1NP, and the markers of bone resorption, CTx and        NTx, also increased albeit to a lesser degree. These results        demonstrate that KRN23 increased bone remodeling which may lead        to an improvement in bone quality in patients with XLH.

The maximum net production of bone mineralization requires the adequatelevels of phosphate consistently over the cycle of dosing in thetreatment of XLH. The present invention is partially based on theobservation that changes in total bound FGF23 levels can occur duringtherapy with KRN23, leading to a complex PK/PD relationship with atypical pattern of waning of the treatment effect during the latter halfof each monthly cycle despite adequate KRN23 drug PK and still remainingPD effect.

The FGF23 production increases during therapy with an anti-FGF23 agentthat can benefit from the management of PK/PD in a manner unexpected byprior data. Although KRN23 monoclonal antibody drug PK data shows that amonthly injection may an adequate therapy, the increased levels ofplasma bound FGF23 levels may provide a decline in the serum phosphatepromoting activity. The serum phosphate pool comprises only 1% of totalbody phosphate, and would likely begin to fall during the latter half ofthe 1 month cycle leading to the potential reduction in net bone formingactivity during the latter half of the dosing cycle. The phosphate levelmight not be declining enough to appreciate the impact of net phosphatebalance in the body because of changing patterns of phosphate recoveryfrom bone induced by the body to support the declining phosphate level.Despite the expected PK predicted plan to dose monthly and to teach awayfrom the use of twice monthly dosing, the PD data showing increasingdecline in phosphate over repeated dosing and the higher bound FGF23production leads to the potential optimize dosing of the anti-FGF23agent to dosing q2 week. This may be particularly advantageous inchildren.

The second change that is occurring is that during the dosing cycle isthe likely decline in phosphate reabsorption which is likely due to lessphosphate transporter expression during the latter half of the monthlydosing cycle. The cyclical pattern of increasing and decreasingtransporter expression would likely lead to a the less efficientcyclical production and then destruction of the Na Pi cotransporter asthe cycle drives up the transporter expression and has the transportergets degraded again during the falling effect of KRN23. During a twicemonthly, more frequent dosing, it is expected that the more efficientconsistent capture of the FGF23 would result in an accumulative neteffect on phosphate reabsorption from increased stable transporterexpression that will be out of proportion to the monthly dose effect onFGF23. By not causing the destruction of the transporter every twoweeks, the transporter protein's own PK might result in the accumulationeffect of the transporter and an out of proportional benefit inphosphate reabsorption. Therefore instead of a proportional PD effect onserum phosphate, the more frequent dosing will result in a cumulativeeffect on NaPi transporter accumulation without decline, and now ahigher achievable phosphate level.

The existing solution of treating FGF23 related disorders is the monthlydosing of a FGF23 antibody (e.g., KRN23) that is based on solid PK datain Phase 1 that would suggest clearly a once per month dose assufficient and it may be an effective dose, especially patients withless phosphate needs like adults. In pediatrics or patients with higherphosphate needs, the increased levels of bound FGF23, and the associatedPD curve of phosphate would be optimally served by a twice monthlydosing interval.

The invention is the novel insight into the unexpected dosing effect attwice monthly rather than monthly. This will be manifested throughincreased and more stable phosphate levels for the same total monthlydose that is out or proportion to the monthly dose effect as determinedby the AUC for serum phosphate and potentially in bone treatmentbenefit.

Example 3: Inhibition of FGF23 with KRN23 Leads to Increased BoneRemodeling

Inventors of the present invention observed a specific effect of theinhibition of FGF23 with an antibody against FGF23 (e.g., KRN23) intreating and reversing the pathologic changes of osteomalacia caused byXLH in adults and children. Treatment with KRN23 corrects the phosphatehomeostasis as was known previously. What is novel and unexpected isthat the inhibition of FGF23 is activating a profound increase in boneremodeling with observed increases in markers of bone formation such asP1NP (serum type 1 pro-collagen/N-terminal) and osteocalcin, alsoincreases marker of bone reabsorption CTX (carboxy-terminal collagencrosslink), see tables 18 to 21 below. Low bone remodeling reflects thehistopathologic changes characteristics of osteomalacia, specificallythe long “mineralization lag time and the increase on osteoid andosteoid wall thickness which explain the lack of osteoclast activity dueto the absence of mineralized bone. While the blocking of FGF23 would beexpected to change renal phosphate reabsorption, 1,25 VitD productionand increased bone mineralization, the clinical data also suggest thatthe increases in bone remodeling (formation and reabsorption) willreverse osteomalacia and restore bone structure, density and improvebone quality preventing the bowing and allow for the normal shaping ofthe skeleton.

Increase in bone formation and reabsorption is the novel additionalmechanism by which the underlying bone pathology in XLH, and inparticular osteomalacia might be healed. This invention also postulatesthat the measurement of markers of bone formation and reabsorption canalso be a way to monitor the regeneration of bone and the replacement ofthe poorly mineralize bone for normal lamellar mineralize bone. Sincethis can be managed actively, the dosing of KRN23 or similar FGF23 agentmight be modulated based on bone resorption and synthesis markers as ascore relevant to bone healing.

Previous treatment of XLH consist of supplementation therapy with oralphosphate and calcitriol or other vitamin D analogs, this treatment mayimprove some aspects of XLH disease, but it does not increase boneremodeling and therefore it is unclear whether it can improveosteomalacia in this patients. Osteomalacia is the reason why adultpatients with XLH have bone pain, micro and stress fracture, delay infracture healing. Biopsy data in our previous research of administratingFGF23 antibody in XLH model mice shows the change from osteomalacia tonormal bone and the change in histomorphometry bone remodeling. See Aonoet al. (Therapeutic Effects of Anti-FGF23 Antibodies in HypophosphatemicRickets/Osteomalacia. J Bone Miner Res. 2009).

The present invention indicates that in order to improve these clinicalcomplications it is necessary to activate bone remodeling. This effecthas never been observed with the current standard of care. Therefore,the present invention provides new methods of treating disorders due toabnormal FGF23 signaling, such as XLH, by activating bone remodeling.For example, according to FIG. 17, In XLH patients, increased FGF23polypeptides lead to decreased serum phosphate and 1,25(OH)₂D, whichresults in decreased bone mineralization (osteomalacia). Osteomalacia inturn leads to decreased osteoclast activity and bone remodeling. As aresult, the patients experience poor bone quality and pain, such as bonedeformities, fractures, and slow fracture healing. Administration ofanti-FGF23 ligand, such as KRN23 will inhibit the FGF23 signaling,normalize serum phosphate and 1,25(OH)₂D, therefore lead to increasedbone mineralization and correction of osteomalacia. As a result,osteoclast and osteoblast activity and bone remodeling are up-regulated,and poor quality bone is replaced with normal lamellar bone, whichultimately leads to restored skeletal health and fracture healing.

TABLE 18 Summary of Bone Biomarkers by Visit/Day: P1NP Efficacy AnalysisP1NP (ng/mL) Change (ng/mL) from Baseline Bone Substudy Bone SubstudyOpen Label Open Label Visit (Day)/ Treatment Total Treatment TotalRelative Day KRN23 KRN23 Placebo KRN23 KRN23 KRN23 Placebo KRN23 Visit 2(D0)/0 N 26 1 1 27 Mean 62.7 100.0 91.0 64.1 Median 65.0 100.0 91.0 69.0Std. Dev. 30.42 NA NA 30.67 Range (Min-Max) (11, (100, (91, ) (11, 123)100) 91 123) Visit 8 (D28)/0 N 26 1 1 27 26 1 1 27 Mean 79.2 93.0 73.079.7 16.5 −7.0 −18.0 15.6 Median 83.0 93.0 73.0 88.0 16.0 −7.0 −18.016.0 Std. Dev. 33.18 NA NA 32.65 15.94 NA NA 16.27 Range (Min-Max) (23,(93, (73, (23, (−13, (−7, (−18, (−13, 157) 93) 73) 157) 54) −7) −18) 54)Visit 14 (D56)/0 N 23 1 1 24 23 1 1 24 Mean 93.5 186.0 66.0 97.4 29.686.0 −25.0 32.0 Median 80.0 186.0 66.0 80.5 21.0 86.0 −25.0 21.5 Std.Dev. 50.71 NA NA 53.06 27.80 NA NA 29.53 Range (Min-Max) (32, (186, (66,(32, (−2, (86, (−25, (−2, 191) 186) 66) 191) 96) 86) −25) 96) Visit 20(D84)/0 N 22 1 1 23 22 1 1 23 Mean 113.9 178.0 67.0 116.7 47.3 78.0−24.0 48.6 Median 112.0 178.0 67.0 115.0 39.0 78.0 −24.0 40.0 Std. Dev.62.17 NA NA 62.19 39.07 NA NA 38.70 Range (Min-Max) (28, (178, (67, (28,(−10, (78, (−24, (−10, 261) 178) 67) 261) 140) 78) −24) 140) End ofStudy - Visit 26 (D120) N 25 1 1 26 25 1 1 26 Mean 119.3 216.0 60.0123.0 55.6 116.0 −31.0 57.9 Median 114.0 216.0 60.0 114.5 41.0 116.0−31.0 43.5 Std. Dev. 74.11 NA NA 75.05 58.86 NA NA 58.88 Range (Min-Max)(24, (216, (60, (24, (−11, (116, (−31, ) (−11, 307) 216) 60) 307) 204)116) −31 204) Early Withdrawal N 1 0 0 1 1 0 0 1 Mean 69.0 69.0 32.032.0 Median 69.0 69.0 32.0 32.0 Std. Dev. NA NA NA NA Range (Min-Max)(69, (69, (32, (32, 69) 69) 32) 32)

TABLE 19 Summary of AUEC_(last) of P1NP Change from Baseline OverallDosing Intervals Efficacy Analysis Set AUEC_(last) Open Label TreatmentBone Substudy Total KRN23 KRN23 Placebo KRN23 Visit 1 through Visit 49 N20 1 0 21 Mean 25373.33 8904.00 24589.07 Median 23998.00 8904.0023494.00 Std. Dev. 19160.505 NA 19018.013 Range (Min-Max) (2854.5,73669.0) (8904.0, 8904.0) (2854.5, 73669.0)

TABLE 20 Summary of Bone Biomarkers by Visit/Day: CTx Efficacy AnalysisCTx (ng/mL) Change (ng/mL) from Baseline Bone Substudy Bone SubstudyOpen Label Open Label Visit (Day)/ Treatment Total Treatment TotalRelative Day KRN23 KRN23 Placebo KRN23 KRN23 KRN23 Placebo KRN23 Visit 2(D0)/0 N 25 1 1 26 Mean 750.1 799.0 613.0 752.0 Median 742.0 799.0 613.0744.0 Std. Dev. 397.36 NA NA 389.45 Range (Min-Max) (214, (799, (613,(214, 1899) 799) 613) 1899) Visit 8 (D28)/0 N 26 1 1 27 25 1 1 26 Mean803.8 623.0 806.0 797.1 24.0 −176.0 193.0 16.3 Median 701.0 623.0 806.0690.0 −5.0 −176.0 193.0 −6.5 Std. Dev. 509.79 NA NA 501.10 160.30 NA NA161.89 Range (Min-Max) (171, (623, (806, (171, (−208, (−176, (193,(−208, 2240) 623) 806) 2240) 471) −176) 193) 471) Visit 14 (D56)/0 N 261 1 27 25 1 1 26 Mean 828.9 653.0 913.0 822.4 76.9 −146.0 300.0 68.3Median 764.0 653.0 913.0 758.0 61.0 −146.0 300.0 58.0 Std. Dev. 444.32NA NA 437.01 168.29 NA NA 170.59 Range (Min-Max) (146, (653, (913, (146,(−170, (−146, (300, (−170 1926) 653) 913) 1926) 481) −146) 300) 481)Visit 20 (D84)/0 N 23 1 1 24 22 1 1 23 Mean 991.6 1030.0 678.0 993.2227.2 231.0 65.0 227.3 Median 900.0 1030.0 678.0 927.0 162.5 231.0 65.0184.0 Std. Dev. 611.08 NA NA 597.70 280.16 NA NA 273.72 Range (Min-Max)(176, (1030, (678, (176, (−133, (231, (65, (−133, 2397) 1030) 678) 2397)927) 231) 65) 927) End of Study - Visit 26 (D120) N 25 1 1 26 24 1 1 25Mean 954.3 774.0 570.0 947.3 187.4 −25.0 −43.0 178.9 Median 859.0 774.0570.0 816.5 108.5 −25.0 −43.0 102.0 Std. Dev. 513.87 NA NA 504.73 261.57NA NA 259.56 Range (Min-Max) (213, (774, (570, (213, (−162, (−25, (−43,(−162, 2170) 774) 570) 2170) 888) −25) −43) 888) Early Withdrawal N 1 00 1 1 0 0 1 Mean 384.0 384.0 −79.0 −79.0 Median 384.0 384.0 −79.0 −79.0Std. Dev. NA NA NA NA Range (Min-Max) (384, (384, (−79, (−79, 384) 384)−79) −79)

TABLE 21 Summary of AUEC_(last) of CTx Change from Baseline OverallDosing Intervals Efficacy Analysis Set AUEC_(last) Open Label TreatmentBone Substudy Total KRN23 KRN23 Placebo KRN23 Visit 1 through Visit 49 N19 1 0 20 Mean 48870.26 −3024.00 46275.55 Median 40327.00 −3024.0039745.00 Std. Dev. 76325.295 NA 75190.381 Range (Min-Max) (−58443.0,222166.0) (−3024.0, −3024.0) (−58443.0, 222166.0)

Example 4: A Randomized, Open-Label, Dose Finding, Phase 2 Study toAssess the Pharmacodynamics and Safety of the Anti-FGF23 Antibody,KRN23, in Pediatric Patients with X-Linked Hypophosphatemia (XLH)

Rationale:

X-linked hypophosphatemia (XLH) is a disorder of renal phosphatewasting, and the most common heritable form of rickets. In XLH patients,high circulating levels of fibroblast growth factor 23 (FGF23) impairnormal phosphate reabsorption in the kidney. Hypophosphatemia andlow-normal circulating 1,25-dihydroxyvitamin D (1,25(OH)₂D) levels aretypical biochemical findings. Low serum phosphorus levels result inhypomineralization of bone and associated abnormalities includingrickets, bowing of the legs, and short stature. The current standard ofcare (SOC) therapy consists of multiple daily doses of oral phosphatecombined with appropriate doses of active vitamin D metabolites. SOCtherapy, when taken with a high degree of compliance and monitoring, canimprove the skeletal disease but often does not fully address the boneand growth abnormalities nor does it target the pathophysiological causeof the disease: renal phosphate wasting induced by high FGF23 levels.SOC therapy also requires careful monitoring to avoid potential riskssuch as nephrocalcinosis, hypercalciuria, and hyperparathyroidism. Moreefficacious, safer, and convenient therapies clearly are needed.

KRN23 is a recombinant fully human monoclonal IgG1 antibody beingdeveloped to treat XLH by binding and inhibiting FGF23 activity, therebyrestoring normal phosphate homeostasis. Three clinical trials have beenconducted in adults with XLH. A Phase 1 study established thepharmacokinetic (PK) profile of KRN23. A Phase 1/2 study and associatedextension study evaluated the pharmacodynamics (PD) of KRN23 onphosphate metabolism and related measures of the phosphate-calciummineral control system. The safety data from these studies has shownthat KRN23 in single and repeated monthly doses up to 1.0 mg/kg was welltolerated by adult XLH subjects. KRN23 sufficiently increased serumphosphorus levels, such that improvements in bone physiology, structureand function would be expected. These data support the initiation offurther studies to evaluate the therapeutic benefit of KRN23 in childrenwho experience the most severe physical and health manifestationsassociated with XLH. Currently, there are no approved treatments and ahigh unmet medical need in pediatric XLH patients.

Adults and children with XLH have the same underlying defect but are ata different stage of the disease. In childhood, normal phosphorus levelsare higher to promote bone formation, whereas in adults, the normalrange is lower coincident with reduced demand for bone formation.Therefore, smaller, more frequent dosing may be preferred for pediatrichypophosphatemic patients to maximize treatment effect without aplateau, drive serum phosphorus levels closer to the normal range andminimize the troughs. This Phase 2 study will examine the PD and safetyof KRN23 administered at multiple doses and dose regimens in pediatricXLH patients.

The study will consist of two periods: a 16-week individual doseTitration Period and a 48-week Treatment Period. The dose response ofKRN23 will be evaluated at 3 starting dose levels. Monthly (Q4) andbiweekly (i.e. every other week; Q2) dosing regimens will also becompared. KRN23 dosing will be individually adjusted every 4 weeks asneeded, according to serum phosphorus levels. The goal is to achievestable serum phosphorus levels in the target range, while minimizingchanges in the calcium control system. Data collected in this study willestablish the dosing strategy, and provide information for the designand endpoint selection for a Phase 3 clinical trial in children withXLH.

Objectives

The objectives of the study are to:

-   -   Identify a dose and dosing regimen of KRN23, based on safety and        PD effect, in pediatric XLH patients    -   Establish the safety profile of KRN23 for the treatment of        children with XLH including ectopic mineralization risk,        cardiovascular effects, and immunogenicity profile    -   Characterize the PK/PD of the KRN23 doses tested in the monthly        (Q4) and biweekly (Q2) dose regimens in pediatric XLH patients    -   Determine the PD effects of KRN23 treatment on markers of bone        health in pediatric XLH patients    -   Obtain a preliminary assessment of the clinical effects of KRN23        on bone health and deformity, muscle strength, and motor        function    -   Obtain a preliminary assessment of the effects of KRN23 on        patient-reported outcomes, including pain, disability, and        quality of life in pediatric XLH patients        Study Design and Methodology:

This study is a randomized, multicenter, open-label, dose finding, Phase2 study. The study will be conducted in prepubescent children aged 5-12years with XLH to assess the PD and safety of KRN23 administered viasubcutaneous (SC) injections monthly (Q4, 28 days±3 days) or biweekly(Q2, 14 days±2 days) for a total of 64 weeks. The study consists of a16-week individual dose Titration Period, followed by a 48-weekTreatment Period. The study will enroll approximately 30 pediatricpatients with XLH and radiographic evidence of bone disease. Subjectswill need to discontinue oral phosphate and vitamin D metabolite therapyprior to randomization and throughout the duration of the study.

There will be 3 cohorts in this study (n=10 per cohort); each with a Q4(n=5) and Q2 (n=5) dosing group. Subjects will be randomized 1:1 to theQ4 or Q2 dosing regimens within each cohort; randomization will bestratified on subject gender. In order to maintain a level of genderbalance, no more than 20 patients of either sex can be enrolled in thestudy. The cohorts will be enrolled sequentially. The first cohort willexamine the lowest doses (0.2 mg/kg Q4 and 0.1 mg/kg Q2) and will beenrolled first. As an added precautionary measure in this pediatricpopulation, the second cohort (0.4 mg/kg Q4 and 0.2 mg/kg Q2) cannotbegin dosing until the fourth subject in the first cohort completes theWeek 4 visit. The third cohort will be administered the highest startingdoses (0.6 mg/kg Q4 and 0.3 mg/kg Q2).

The initial 16-week Titration Period is intended to identify the KRN23dose required to achieve the target peak PD effect. The goal is toidentify an individualized KRN23 dose which maintains serum phosphoruslevels in the target range, however the dose level should not exceed 2.0mg/kg for the Q4 regimen and 1.0 mg/kg for the Q2 regimen. The targetfasting serum phosphorus range for this study is 3.5-4.5 mg/dL(1.13-1.45 mmol/L), based on the peak PD effect of KRN23.

The dose will be adjusted every 4 weeks, as needed, based on 2-weekpost-dose (peak) fasting serum phosphorus levels. The KRN23 dosetitration scheme (Table 22) will be used as a guideline should the peakfasting serum phosphorus level fall outside of the target range. If theserum phosphorus level is rising but has not yet reached the acceptabletarget range by the end of the Titration Period, the titration cancontinue into the Treatment Period until the target range is reachedprovided there are no safety concerns.

TABLE 22 KRN23 Dose Titration Scheme Serum Phosphorus (2 weeksPost-Dose) Dose Adjustment ¹ <3.5 mg/dL ² In 2 weeks, increase dose by0.1 mg/kg for <1.13 mmol/L Q2 OR 0.2 mg/kg for Q4  3.5-4.5 mg/dL Repeatprevious dose 1.13-1.45 mmol/L >4.5 mg/dL (1.45 mmol/L) In 2 weeks,decrease dose by 0.1 mg/kg for and ≤ age adjusted ULN Q2 OR 0.2 mg/kgfor Q4 > age adjusted ULN Skip next 2 doses for Q2 OR skip next dose forQ4, then re-initiate dosing at last dose level ¹ Dose adjustments forsubjects assigned to the Q2 regimen will only be made after 2consecutive peak measurements. ² If a subject's serum phosphorus levelhas not increased, as defined by a change no greater than 0.1 mg/dL,after 2 consecutive dose escalations, even if the target range has notbeen achieved, then the previous dose will be considered that subject'soptimized dose and not escalated further.

At the end of the Titration Period, the population of 30 subjects willconsist of essentially two groups of 15 subjects, each with individuallyoptimized dosing of KRN23 at either a Q4 week or Q2 week frequency. Ananalysis of safety and select PD data is planned at the end of theTitration Period (Week 16). A second analysis is planned following thefirst 24-weeks of the Treatment Period (Week 40) to compare treatmentoutcomes to baseline (pre-dose). FIG. 18 provides a schematic of theoverall study design.

Number of Subjects:

Approximately 30 pediatric subjects will be enrolled in the study.Subjects who withdraw or are removed from the study may be replaced on acase-by-case basis.

Diagnosis and Criteria for Inclusion and Exclusion:

Individuals eligible to participate in this study must meet all of thefollowing criteria:

1) Male or female, aged 5-12 years, inclusive, with open growth plates

2) Tanner stage of 2 or less based on breast and testicular development(assessed only in children ≥8 years of age)

3) Diagnosis of XLH supported by ONE of the following:

-   -   Confirmed PHEX mutation in the patient or a directly related        family member with appropriate X-linked inheritance    -   Serum iFGF23 level ≥30 pg/mL by Kainos assay        4) Biochemical findings associated with XLH including:    -   Serum phosphorus ≤2.8 mg/dL (0.904 mmol/L)*    -   Serum creatinine within age-adjusted normal range*        5) Short stature as defined by standing height <25% percentile        for age and gender (per CDC 2000)        6) Radiographic evidence of active bone disease including        rickets in the wrists and/or knees, AND/OR femoral/tibial bowing        7) Willing to provide access to prior medical records for the        collection of historical growth, biochemical and radiographic        data, and disease history.        8) Provide written or verbal assent (if possible) and written        informed consent by a legally authorized representative after        the nature of the study has been explained, and prior to any        research-related procedures        9) Must, in the opinion of the investigator, be willing and able        to complete all aspects of the study, adhere to the study visit        schedule and comply with the assessments.        Individuals who meet any of the following exclusion criteria        will not be eligible to participate in the study:        1) Use of a pharmacologic vitamin D metabolite or analog (e.g.        calcitriol, doxercalciferol, and paricalcitol) within 14 days        prior to Screening Visit 2; washout will take place during the        Screening Period        2) Use of oral phosphate within 7 days prior to Screening Visit        2; washout will take place during the Screening Period        3) Use of aluminum hydroxide antacids (e.g. Maalox® and        Mylanta®), systemic corticosteroids, and thiazides within 7 days        prior to Screening Visit 1        4) Use of growth hormone within 1 year prior to Screening Visit        1        5) Use of bisphosphonates for 6 months or more in the 2 years        prior to Screening Visit 1        6) Presence of nephrocalcinosis on renal ultrasound graded ≥3        based on the following scale:        0=Normal        1=Faint hyperechogenic rim around the medullary pyramids        2=More intense echogenic rim with echoes faintly filling the        entire pyramid        3=Uniformly intense echoes throughout the pyramid        4=Stone formation: solitary focus of echoes at the tip of the        pyramid        7) Planned or recommended orthopedic surgery, including staples,        8-plates or osteotomy, within the clinical trial period        8) Hypocalcemia or hypercalcemia, defined as serum calcium        levels outside the age-adjusted normal limits*        9) Evidence of tertiary hyperparathyroidism as determined by the        Investigator        10) Use of medication to suppress PTH (e.g. Sensipar®,        cinacalcet) within 2 months prior to Screening Visit 1        11) Presence or history of any condition that, in the view of        the investigator, places the subject at high risk of poor        treatment compliance or of not completing the study.        12) Presence of a concurrent disease or condition that would        interfere with study participation or affect safety        13) Positive for human immunodeficiency virus antibody,        hepatitis B surface antigen, and/or hepatitis C antibody        14) History of recurrent infection or predisposition to        infection, or of known immunodeficiency        15) Use of a therapeutic monoclonal antibody within 90 days        prior to Screening Visit 1 or history of allergic or        anaphylactic reactions to any monoclonal antibody        16) Presence or history of any hypersensitivity to KRN23        excipients that, in the judgment of the investigator, places the        subject at increased risk for adverse effects.        17) Use of any investigational product or investigational        medical device within 30 days prior to screening, or requirement        for any investigational agent prior to completion of all        scheduled study assessments.        * Criteria to be determined based on overnight fasting (min. 8        hours) values collected at Screening Visit 2

Investigational Product, Dose and Mode of Administration: KRN23 is asterile, clear, colorless, and preservative-free solution in single-use5-mL vials containing 1 mL of KRN23 at a concentration of 10 mg/mL or 30mg/mL. Subjects will receive study drug via SC injection to the abdomen,upper arms and thighs; the injection site will be rotated with eachinjection. Subjects will be sequentially enrolled into the cohorts,starting with the lowest dose group, and randomized to a dosing regimen(Q2 or Q4) (FIG. 2.1) then individually titrated to achieve a targetovernight fasting serum phosphorus range of 3.5-4.5 mg/dL (1.13-1.45mmol/L). The maximum dose allowed in this protocol is 2.0 mg/kg for theQ4 regimen, and 1.0 mg/kg for the Q2 regimen.

Reference Therapy, Dose and Mode of Administration: The study design isopen-label; all subjects will receive investigational product. Noplacebo or reference therapy will be administered in this study.

Duration of Treatment: The study consists of two periods: a 16-weekindividual dose Titration Period, followed by a 48-week TreatmentPeriod. The planned duration of treatment in this study is 64 weeks.

Criteria for Evaluation:

Pharmacodynamic*:

-   -   Serum phosphorus    -   Serum 1,25(OH)2D    -   Urinary phosphorus    -   Phosphate reabsorption: ratio of renal tubular maximum        reabsorption rate of phosphate to glomerular filtration rate        (TmP/GFR), and tubular reabsorption of phosphate (TRP)    -   Bone biomarkers: procollagen type 1 N-propeptide (P1NP),        carboxy-terminal cross-linked telopeptide of type I collagen        (CTx), and alkaline phosphatase (ALP)

Blood and urine to be collected after a minimum overnight fasting timeof 8 hours and prior to drug administration (if applicable) per dosingregimen.

Efficacy—Bone Health:

-   -   Growth: standing height, sitting height, arm length and leg        length will be measured. Growth percentiles based on standing        height will be derived prior to and following treatment if        historical data are available    -   Severity of rickets and epiphyseal (growth plate) abnormalities:        central readings of bilateral posteroanterior (PA) hand/wrist        and anteroposterior (AP) knee radiographs using a        disease-specific qualitative Radiograph Global Impression of        Change (RGI-C) scoring system and a modified version of a scale        developed for nutritional rickets    -   Lower extremity deformity assessed by intercondylar distance and        intermalleolar distance. Specific abnormalities related to lower        extremity deformity and bowing observed on standing long leg        films will also be evaluated using the qualitative RGI-C scoring        system    -   Bone Mineral Density or Content at the cortical and trabecular        compartment as assessed by XtremeCT of the forearm and tibia        (performed at select sites depending on availability of        equipment)

Efficacy—Clinical Outcomes:

-   -   Walking ability: Six Minute Walk Test (6MWT) total distance and        percent of predicted normal    -   Gross motor function: Bruininks-Oseretsky Test of Motor        Proficiency—Second Edition (BOT-2) subtests to assess running        speed, agility and strength    -   Muscle strength: bilateral hand-held dynamometry (HHD) in the        following muscle groups: gross grip, knee flexors, knee        extensors, hip flexors, hip extensors and hip abductors    -   Functional disability and pain: Pediatric Orthopedic Society of        North America Pediatric Outcomes Data Collection Instrument        (POSNA PODCI)    -   Health-Related Quality of Life: SF-10 for Children Health Survey        (SF-10)

Pharmacokinetic:

-   -   Serum KRN23 (pre-dose level)

Safety Assessments: Safety will be evaluated by the incidence, frequencyand severity of adverse events (AEs) and serious adverse events (SAEs),including clinically significant changes from baseline to scheduled timepoints. General safety variables include:

-   -   Vital signs and weight    -   Interval history and physical examinations    -   GFR    -   Chemistry, hematology, and urinalysis, including additional        KRN23/XLH biochemical parameters of interest (serum 25(OH)D,        amylase, creatinine, and iFGF23 [total, bound and unbound])    -   Anti-KRN23 antibody testing and dose-limiting toxicities    -   Concomitant medications

Ectopic mineralization safety assessments include:

-   -   Renal ultrasound    -   ECHO and ECG    -   Serum calcium, phosphorus and iPTH; urinary calcium and        creatinine

Data Monitoring Committee (DMC): An independent DMC that includesmembers with expertise in metabolic bone disease and the conduct ofclinical trials in children will act in an advisory capacity to monitorsubject safety on a routine basis throughout the trial. The DMC willalso meet for quarterly data reviews.

Statistical Methods: A full description of the statistical evaluationswill be provided in the Statistical Analysis Plan.

Sample Size: A sample size of 10 per cohort will provide at least 90%power to detect a serum phosphorus increase from baseline of at least0.8 mg/dL, assuming a standard deviation of 0.7 mg/dL or smaller, at the2-sided level of significance of 0.05. In addition, a total sample sizeof 30 subjects (15 subjects per Q4 or Q2 regimen) will provide at least90% power to detect a 0.5 mg/dL difference between the two dosingregimens assuming a standard deviation of 0.4 and 2-sided level ofsignificance of 0.05. Phosphate and mineral control are adequatelypowered based on the clinical experience to date with KRN23. The degreeof powering for bone health will depend on the degree of effect expectedwhich is not known. However, powering for adequate phosphate controlshould provide the potential for improved bone health based on priorexperience with oral phosphate replacement therapy.

Pharmacodynamics and Efficacy Analysis: Analyses of PD and efficacycomparing the two dosing regimens will be performed at Week 40 and Week64 with Week 0 as the baseline. In addition, PD and safety data will besummarized for each cohort and each dose regimen within a cohort aftereach cohort has completed the Titration Period (Week 16). Descriptivestatistics will be used to summarize the data. For continuous variables,the mean, standard error, median, minimum, and maximum will be provided.For discrete data, the frequency and percent distributions will beprovided. Changes over time and the association of the efficacy with thePD variables will be summarized and evaluated.

Safety Analysis: All subjects who receive any amount of study drug willbe included in the safety analysis. Safety of each cohort and each doseregimen within a cohort will be assessed.

The multiple-dose, dose escalation Phase 1/2 study of Example 1 wasconducted in adult XLH subjects. KRN23 was well tolerated following SCadministration of 4 intra-subject escalating doses (0.05 mg/kg→0.1mg/kg→0.3 mg/kg→0.6 mg/kg) administered once per 28 days. The proportionof KRN23-treated subjects with serum phosphorus levels in the targetrange (>2.5 to ≤3.5 mg/dL) increased with KRN23 dose level but did notexceed the upper limit of normal (4.5 mg/dL) in any subject at any timepoint. A direct PK-PD relationship between serum KRN23 concentrationsand serum phosphorus levels was noted in the study. In an associatedextension study of Example 2, doses up to 1.0 mg/kg KRN23 administeredmonthly were well tolerated by adult XLH subjects over a period of 48weeks.

Adults and children with XLH have the same underlying defect but are ata different stage of the disease. In childhood, normal phosphorus levelsare higher to promote bone formation, whereas in adults, the normalrange is lower coincident with reduced demand for bone formation.Successful treatment of XLH requires sustained increases in serumphosphorus levels (Carpenter et al. 2011). Smaller, more frequent dosingmay be preferred for pediatric hypophosphatemic patients to maximizetreatment effect by maintaining serum phosphorus levels closer to thenormal range and minimize the troughs. Therefore, the key objectives ofthis study are to determine both the optimal KRN23 dosing regimen forpediatric XLH patients and identify an acceptable dose that will allowfor the improvement in rickets and associated clinical consequences,while avoiding hypercalciuria, hypercalcemia and hyperparathyroidism.

The dose-finding goal of this study is to identify an individualizedKRN23 dose which maintains serum phosphorus levels in the target range.Dose finding will be conducted progressively in three distinct dosingcohorts, each evaluating a different starting dose level and doseregimen (Q4 or Q2). Starting doses for all three cohorts are below thehighest doses studied in adult XLH (1 mg/kg administered monthly). Doseescalation will be individually titrated based on PD effects on serumphosphorus, safety and tolerability. Dosing will be initiated andtitrated in a stepwise, controlled fashion over a 16-week period toachieve serum phosphorus levels in a target range below the age-adjustedupper limit of normal.

Since phosphorus requirements in growing children are greater than inadults, the design provides the provision to increase the dose level ashigh as 2.0 mg/kg for the Q4 regimen and 1.0 mg/kg for the Q2 regimen.Previous studies with KRN23 in adult XLH patients did not show any “offtarget” effects, therefore the safety profile is expected to be relatedsolely to the PD effect, essentially increased serum phosphorus. Sincethe serum phosphorus target range is defined well below the upper limitof normal, the likelihood of a dose-related safety issue is low. Theefficacy data on phosphate control from these previous studies alsosuggest there is a plateau in effect between 0.6-1.0 mg/kg which may ormay not be the case in pediatric patients in whom phosphate metabolismis clearly different. Therefore, the protocol allows limited flexibilityto adapt to incrementally higher doses, if the expected doses based onadult data are inadequate to achieve an acceptable increase in serumphosphorus within the accepted safe range.

The target fasting serum phosphorus range for this study is 3.5-4.5mg/dL (1.13-1.45 mmol/L), based on the peak PD effect of KRN23, which isapproximately 14 days post-dose. The target range represents the low tomid-range of normal values in children. This level is sufficient toimprove rickets and other bone defects, while minimizing the risk ofectopic mineralization. The dose will be adjusted every 4 weeks, asneeded, based on 2-week post-dose (peak) fasting serum phosphorus levelsaccording to the titration scheme detailed below. The titration scheme(Table 23) will be used as a guideline for dose adjustments should thepeak fasting serum phosphorus level fall outside of the target range. Ifthe serum phosphorus level is rising but has not yet reached theacceptable target range by the end of the Titration Period, thetitration can continue into the Treatment Period until the target rangeis reached provided there are no safety concerns.

TABLE 23 KRN23 Dose Titration Scheme Serum Phosphorus (2 weeksPost-Dose) Dose Adjustment ¹ <3.5 mg/dL ² In 2 weeks, increase dose by0.1 mg/kg for <1.13 mmol/L Q2 OR 0.2 mg/kg for Q4  3.5-4.5 mg/dL Repeatprevious dose 1.13-1.45 mmol/L >4.5 mg/dL (1.45 mmol/L) In 2 weeks,decrease dose by 0.1 mg/kg for and ≤ age adjusted ULN Q2 OR 0.2 mg/kgfor Q4 > age adjusted ULN Skip next 2 doses for Q2 OR skip next dose forQ4, then re-initiate dosing at last dose level ¹ Dose adjustments forsubjects assigned to the Q2 regimen will only be made after 2consecutive peak measurements. ² If a subject's serum phosphorus levelhas not increased, as defined by a change no greater than 0.1 mg/dL,after 2 consecutive dose escalations, even if the target range has notbeen achieved, then the previous dose will be considered that subject'soptimized dose and not escalated further.

The planned duration of treatment in this study is 64 weeks. The studyconsists of two periods: a 16-week individual dose Titration Period,followed by a 48-week Treatment Period.

Example 5: Preliminary Results from Pediatric Phase 2 Study

This example illustrates that Q2W dosing of KRN23 provides stable andsteady increases in serum phosphorus levels, while a Q4W dosing regimenproduces larger serum phosphorus peaks and troughs.

In this example, there were 18 XLH patients in the Q2W treatment group,with 3 cohorts: (1) with a starting dose of 0.1 mg/kg, (2) with astarting dose of 0.2 mg/kg, and (3) with a starting dose of 0.3 mg/kg.There were also 18 patients in the Q4W treatment group, with 3 cohorts:(1) with a starting dose of 0.2 mg/kg, (2) with a starting dose of 0.4mg/kg, and (3) with a starting dose of 0.6 mg/kg. Patients ranged in agefrom 5-12 years, with a mean age of 8.2 years.

As noted in example 4, the planned duration of the study is 64 weeks andconsists of two periods: a 16-week individual dose Titration Period,followed by a 48-week Treatment Period. Serum phosphorus levels in theQ2W treatment group through 16 weeks of treatment is shown in FIG. 19,which illustrates a relatively steady and stable increase in serumphosphorus levels during 16 weeks of treatment with KRN23. By contrast,serum phosphorus levels in the Q4W treatment group showed larger peaksand troughs, as illustrated in FIG. 20. A side-by-side comparison ofserum phosphorus levels in the Q2W treatment group versus the Q4Wtreatment group across all cohorts is illustrated in FIG. 21. Anumerical illustration of serum phosphorus levels in the Q2W and Q4Wtreatment groups through 24 weeks is shown in Table 24 below.

TABLE 24 Serum Phosphorus Levels by Regimen - Overall Regimen W0 W14 W16W22 W24 N Q2 18 17 18 11 9 Q4 18 17 17 12 10 Mean serum P; Q2 2.4863.071 3.050 3.218 3.400 mg/dL Q4 2.272 3.300 2.529 3.325 2.830 Meanserum P; Q2 — 0.571 0.567 0.673 0.700 Δ vs. BL Q4 — 1.012 0.229 1.0330.460 In target range; Q2 — 5 (29.4%) 9 (50%) 6 (54.5%) 7 (77.8%) N (%)Q4 — 12 (70.6%) 0 (0%) 9 (75.0%) 2 (20.0%)

Similar to the observations with serum phosphorus levels, TmP/GFR levelsin the Q2W treatment group also increased at a steadier and more stablepace in comparison to the Q4W treatment group. TmP/GFR levels in the Q2Wtreatment group through 16 weeks of treatment is shown in FIG. 22, whileTmP/GFR levels in the Q4W treatment group are shown in FIG. 23. Aside-by-side comparison of TmP/GFR levels in the Q2W treatment groupversus the Q4W treatment group across all cohorts is illustrated in FIG.24. A numerical illustration of serum phosphorus levels in the Q2W andQ4W treatment groups through 24 weeks is shown in Table 25 below.

TABLE 25 TmP/GFR Levels by Regimen-Overall Regimen W0 W14 W16 W24 N Q218 17 18 7 Q4 17 16 16 8 Mean Tmp/GFR; Q2 2.249 3.136 3.126 3.479 mg/dLQ4 1.987 3.418 2.298 2.644 Mean Tmp/GFR; Q2 — 0.864 0.876 1.054 Δ vs. BLQ4 — 1.327 0.249 0.503

Levels of 1,25(OH)₂D followed a similar profile as serum phosphorus. Anumerical illustration of 1,25(OH)₂D levels in the Q2W and Q4W treatmentgroups through 28 weeks is shown in Table 26 below.

TABLE 26 1,25(OH)₂D Levels by Regimen-Overall Regimen W0 W14 W16 W28 NQ2 18 17 18 7 Q4 17 16 16 8 Mean 1,25(OH)₂D; Q2 40.083 65.607 59.77686.460 pg/mL Q4 40.578 77.775 50.639 47.933

Levels of the bone biomarker serum alkaline phosphatase (ALP) were alsomeasured and showed a decrease over time for both treating regimens, asillustrated in FIG. 25.

In terms of safety, no severe adverse events were observed through 16weeks of treatment and there were no adverse events leading todiscontinuation. This suggests KRN23 is safe and well-tolerated throughthe first 16 weeks of the Q2W and Q4W dosing regimens. There were alsono observable changes in serum and urinary calcium levels, and minimalchanges in mean iPTH, similar to those observed in adults. Lastly, FGF23levels were seen to increase over the course of KRN23 treatment, asillustrated in Table 27 below.

TABLE 27 iFGF23 Levels by Regimen-Overall Regimen Baseline W8 W16 W28 NQ2 18 18 18 4 Q4 18 18 18 5 Mean iFGF23; Q2 163.6 144655.6 231368.9314650.0 pg/mL Q4 165.7 101815.6 149015.0 131994.0

In conclusion, the dose response in serum phosphorus levels appears tobe similar in adult and pediatric patients, with noticeable peaks andtroughs observed in the Q4W regimen in comparison to a much steadier andmore stable increase in the Q2W regimen. The results in this examplefurther indicate an increase of approximately 1 mg/dL in serumphosphorus levels in response to 1 mg/kg of the KRN23 therapeutic.Additionally, TmP/GFR and 1,25(OH)₂D levels generally followed the sameprofile as serum phosphorus levels in response to Q2W and Q4W dosing,with more stable and steady increases over time in the Q2W dosingtreatment group.

The disclosures, including the claims, figures and/or drawings, of eachand every patent, patent application, and publication cited herein arehereby incorporated herein by reference in their entireties.

Unless defined otherwise, all technical and scientific terms herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materials,similar or equivalent to those described herein, can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein. All publications, patents, and patentpublications cited are incorporated by reference herein in theirentirety for all purposes.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

What is claimed:
 1. A method of treating X-linked hypophosphatemia(XLH), comprising administering to a subject in need of such treatmentan effective amount of an anti-FGF23 antibody, wherein the anti-FGF23antibody is administered subcutaneously about every two weeks, andwherein the anti-FGF23 antibody comprises the CDR sequences of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQID NO:
 6. 2. The method of claim 1, wherein the heavy chain of theanti-FGF23 antibody comprises a sequence of SEQ ID NO:
 7. 3. The methodof claim 1, wherein the light chain of the anti-FGF23 antibody comprisesa sequence of SEQ ID NO:
 8. 4. The method of claim 1, wherein the heavychain of the anti-FGF23 antibody comprises a sequence of SEQ ID NO: 7and the light chain of the anti-FGF23 antibody comprises a sequence ofSEQ ID NO:
 8. 5. The method of claim 1, wherein the anti-FGF23 antibodyis administered with a pharmaceutically-acceptable carrier.
 6. Themethod of claim 1, wherein the subject is a pediatric subject.
 7. Themethod of claim 1, wherein the anti-FGF23 antibody is administered at adose from about 0.05 mg/kg to about 1.0 mg/kg.
 8. The method of claim 1,wherein the anti-FGF23 antibody is administered at a dose of about 0.8mg/kg.
 9. The method of claim 1, wherein the anti-FGF23 antibody isadministered at a dose of about 1.0 mg/kg.